EP2545187A1 - Methods of diagnosing and treating cancer in patients having or developing resistance to a first cancer therapy - Google Patents

Methods of diagnosing and treating cancer in patients having or developing resistance to a first cancer therapy

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Publication number
EP2545187A1
EP2545187A1 EP11709278A EP11709278A EP2545187A1 EP 2545187 A1 EP2545187 A1 EP 2545187A1 EP 11709278 A EP11709278 A EP 11709278A EP 11709278 A EP11709278 A EP 11709278A EP 2545187 A1 EP2545187 A1 EP 2545187A1
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Prior art keywords
inhibitor
raf
cancer
cot
kinase
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German (de)
French (fr)
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EP2545187B1 (en
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Levi A. Garraway
Cory M. Johannessen
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Dana Farber Cancer Institute Inc
Broad Institute Inc
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Dana Farber Cancer Institute Inc
Broad Institute Inc
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4184—1,3-Diazoles condensed with carbocyclic rings, e.g. benzimidazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/11—Protein-serine/threonine kinases (2.7.11)
    • C12Y207/11024—Mitogen-activated protein kinase (2.7.11.24), i.e. MAPK or MAPK2 or c-Jun N-terminal kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • BRAF serine/threonine kinase B-RAF
  • MAPK mitogen-activated protein kinase
  • the present invention relates to the development of resistance to therapeutic agents in the treatment of cancer and identification of targets that confer resistance to treatment of cancer.
  • the present invention also relates to identification of parallel drug targets for facilitating an effective long-term treatment strategy and to identifying patients that would benefit from such treatment.
  • a method of identifying a subject having cancer who is likely to benefit from treatment with a combination therapy with a RAF inhibitor and a second inhibitor includes assaying a gene copy number, a mRNA or a protein level or phosphorylation of one or more kinase targets selected from the group consisting of MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL(CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3) in cancer cells obtained from the subject.
  • MAP3K8 TPL2/COT
  • RAF1 CRAF
  • CrkL CrkL
  • FGR Fgr
  • PRKCE PRKCE
  • Prkch PRKCH
  • ErbB2 ErbB2
  • AXL Axl
  • PAK3 PAK3
  • the method further includes comparing the gene copy number, the mRNA or the protein level or the phoshorylation with a gene copy number, a mRNA or a protein level or phosphorylation of the target kinase in cells obtained from a subject without the cancer and correlating increased gene copy number or an alteration in mRNA expression or protein overexpression or phosphorylation of the target kinase in the cancer cells relative to the cells from the subject without the cancer with the subject having the cancer who is likely to benefit from treatment with the combination therapy.
  • a method of treating cancer in a subject in need thereof includes administering to the subject an effective amount of a RAF inhibitor and an effective amount of a second inhibitor, wherein the second inhibitor is a MEK inhibitor or a TPL2/COT inhibitor.
  • a method of identifying a kinase target that confers resistance to a first inhibitor includes culturing cells having sensitivity to the first inhibitor and expressing a plurality of kinase ORF clones in the cell cultures, each cell culture expressing a different kinase ORF clone.
  • the method further includes exposing each cell culture to the inhibitor and identifying cell cultures having greater viability than a control cell culture after exposure to the inhibitor to identify the kinase ORF clone that confers resistance to the first inhibitor.
  • Figure 1 illustrates an ORF-based functional screen which identified COT and C-RAF kinases as drivers of resistance to B-RAF inhibition,
  • Figure 2 illustrates resistance to B-RAF inhibition via MAPK pathway activation
  • (a) Indicated ORFs were expressed in A375. Levels of phosphorylated MEK and ERK were assayed following 18 h. treatment with DMSO (-) or PLX4720
  • COT mRNA has an internal start codon (30M) resulting in two protein products of different lengths; amino acids 1 -467 or 30-467, noted with arrows, (e) COT and ERK phosphorylation in lysates from A375 expressing indicated ORFs following shRNA-mediated B-RAF depletion (shBRAF) relative to control shRNA (shLuc). (f) ERK phosphorylation in lysates from A375 expressing indicated ORFs following
  • shRNA-mediated C-RAF depletion (shCRAF) or control shRNA (shLuc), following 18 h. treatment with DMSO (-) or 1 ⁇ PLX4720 (+).
  • Figure 3 illustrates COT expression predicts resistance to B-RAF inhibition in cancer cell lines, (a) MAP3K8/COT copy number; red bars: COT amplification, blue bars: non-amplified COT; (b) COT expression in B-RAF V600E cell lines and (c) short-term cultures; (d) PLX4720 GI50 in B-RAF V600E cell lines.
  • OUMS-23 and M307 represent cell lines with COT expression/amplification and all others represent cell lines with undetectable/unaltered COT;
  • FIG. 4 illustrates COT-expressing B-RAFV600E cell lines exhibit resistance to allosteric MEK inhibitors, (a) CI-1040 GI50 in a panel of B-RAF V600E cell lines; (b) MEK and ERK phosphorylation in lysates from indicated cell lines treated with DMSO or CI-1040 (concentration noted); (c) Fold change (relative to MEK1 ) GI50 of A375 ectopically expressing the indicated ORFs for PLX4720, RAF265, CI-1040 and AZD6244; (d) ERK phosphorylation in A375 expressing indicated ORFs following treatment with DMSO or 1 ⁇ of PLX4720, RAF265, CI-1040 or AZD6244; (e) Viability of A375 expressing the indicated ORFs and treated with DMSO, PLX4720 (concentration indicated) and PLX4720 in combination with CI-1040 or AZD6244 (all 1 ⁇
  • PLX4720 in combination with CI-1040 or AZD6244 (all 1 ⁇ ); (g) Cell lines with aberrant MAP3K8/COT copy number/expression are insensitive to the allosteric MEK inhibitor CI-1040 or (h) AZD6244; (i) A schematic outlining the formation of MAP3K complexes in response to B-RAF inhibition in B-RAF V600E -mutant cell lines.
  • PLX4720 positions C-RAF in a signaling-competent complex (upper right panel) that is activated by oncogenic events upstream of C-RAF (lower right panel), subsequently driving resistance. In the context of COT expression, COT/RAF-containing complexes are sufficient to activate the MAPK pathway and mediate resistance (lower left panel).
  • Figure 5 illustrates a schematic outline of an ORF-based functional screen for kinases that drive resistance to B-RAF inhibition.
  • the B-RAF V600E cell line A375 was lentivirally transduced with the 597 kinases in the CCSB/Broad Institute Kinase ORF Collection.
  • ORFs having a positive or negative effect on proliferation in control-treated A375 were identified and removed from final analysis.
  • Resistance-promoting ORFs were identified by generating a differential viability ratio between B-RAF inhibited (PLX4720-treated) and control-treated cells. Differential viability was normalized to a constitutively active MEK1 allele, MEK1 DD ; an assay specific positive control.
  • FIG. 6 illustrates that the CCSB/Broad Institute Kinase ORF Collection is well expressed via high titer lentivirus.
  • (a) schematic of the pLX-BLAST-V5 lentiviral expression vector used for all ORF-screens and subsequent validation
  • (b) GFP-tagged ORFs representing a broad size range were lentivirally expressed in Jurkat cells and the percentage of GFP-expressing cells/ORF (e.g., infected cells) quantified, demonstrating high viral titer across a range of ORF sizes
  • Figure 7 illustrates the expression of candidate resistance ORFs.
  • 293T were transiently transfected with pLX-BLAST-V5-ORF (indicated) and expression detected using an anti-V5-HRP antibody.
  • the AXL clone is 'closed' and has a stop codon preceding the V5 tag. See Figure 12 for verification of expression; ( * ) on dark-exposure indicate the expression of three ORFs not visible in the lighter exposure.
  • Figure 8 illustrates that a secondary screen prioritizes the top 9 candidate B-
  • RAF inhibitor resistance ORFs The top nine ORFs scoring in the primary screen were expressed in A375 or SKMEL28 and a Gl 50 from an 8-point PLX4720 concentration range.
  • Figure 9 illustrates the effects of ORF expression on proliferation in B- RAF V600E cell lines. Proliferation, relative to MEK1 , in (a) A375 or (b) SKMEL28 expressing indicated ORFs after 7 days of growth.
  • FIG. 10 illustrates that ectopic expression of constitutively active MEK1 (MEK1 DD ) and COT lead to increased pMEK/pERK in A375, whereas C-RAF reduces pMEK/pERK levels.
  • Lysates from A375 ectopically expressing GFP, MEK1 , MEK DD , COT or C-RAF were analyzed via immunoblot for levels of pERK and pMEK.
  • GFP and MEK1 (lanes 1 -3) were separated from COT/C-RAF (lanes 4-5) to prevent residual V5- MEK1 signal from overwhelming that of COT and C-RAF, which are expressed at much lower levels.
  • FIG 11 illustrates that the kinase activity of COT and C-RAF is required for sustained ERK phosphorylation in the context of PLX4720 treatment.
  • Immunoblot analysis of A375 expressing ectopic (a) MEK1 , wild type COT or kinase inactive COT (COT K167R ) or (b) MEK1 , wild type C-RAF or kinase inactive C-RAF (C-RAF K375M ) treated with 1 ⁇ PLX4720 for 18 h.
  • Figure 12 illustrates the effects of ORF expression on MAPK signaling in the context of the B-RAF inhibitor PLX4720.
  • MAPK pathway activation was assessed by immunoblot analysis of pERK and pMEK in A375 expressing the indicated ORFs in the presence of PLX4720 (18 h., concentration indicated).
  • ( * ) indicates the use of an antibody directed against the expressed ORF, not the V5 epitope.
  • AXL was cloned without the V5 tag.
  • FIG 13 illustrates that B-RAF associates with immunoprecipitated C-RAF in A375 following 18 hr. treatment with 1 ⁇ PLX4720 (+) or DMSO (-), (a). WCE; whole cell extract controls. Ectopically expressed C-RAF constitutively associates with B-RAF and is phosphorylated at S338, consistent with membrane localization and activation. MEK1 , MEK DD and COT-expressing A375 show no evidence of C-RAF activation, (b).
  • FIG 14 illustrates that Retroviral expression of a wild-type C-RAF or a high-activity truncation mutant of C-RAF (C-RAF(22W)) renders A375 resistant to the B- inhibitor PLX4720 (a) and leads to sustained pERK levels in the context of PLX4720 treatment (1 ⁇ , 18 h.), (b).
  • C-RAF expression levels achieved with retroviruses are significantly lower than with lentiviral-based systems, resulting in a lower GI 5 o than that achieved with lentiviral C-RAF.
  • Figure 15 illustrates the effects of B-RAF V600E on COT mRNA
  • Reverse-transcribed mRNA was analyzed for GAPDH- normalized COT expression, relative to GFP-expressing, DMSO treated, A375. ( * ) Not significant, p > 0.05 (Student's two-tailed, paired T-Test). Error bars represent SEM.
  • FIG. 16 illustrates that B- and C-RAF protein levels are not required for COT-mediated ERK phosphorylation.
  • A375 expressing ectopic MEK1 (control) or COT were sequentially infected with lentivirus expressing shRNAs targeting B-RAF, C-RAF or control shRNA (shLuc) and assayed for expression of the indicated proteins in the presence (+) or absence (-) of 1 ⁇ PLX4720, 18 h.
  • Figure 17 illustrates SNP analysis of 752 cell lines reveals copy number alterations in MAP3K8/COT. Of the 752 cell lines hat had undergone copy number analysis, 534 had also undergone mutation profiling. Thirty-eight (7.1 %) of mutation- profiled cells harbor the B-RAF V600E mutation. Two cell lines (OUMS-23, RPMI-7951 , indicated) harbor the B-RAF V600E mutation along with copy number gain in
  • Figure 18 illustrates MAP3K8/COT alterations in the cancer cell line OUMS- 23.
  • OUMS-23 is one of the top 2% (of 765 cell lines) expressing COT mRNA.
  • Figure 19 illustrates that COT mRNA and protein are expressed in B RAF- inhibitor resistant cell lines and tissue, (a) RT/PCR analysis of GAPDH normalized COT mRNA expression in a panel of cell lines, short term cultures and tissue from relapsed, PLX4032-treated, malignant melanoma (MM-R).
  • Figure 20 illustrates that depletion of COT affects viability in the COT amplified cell line RPMI-7951 .
  • Figure 21 illustrates effects of ORF expression on the GI 5 o of a panel of MAPK pathway inhibitors in SKMEL28.
  • FIG. 22 illustrates that COT can activate ERK via MEK-independent and MEK-dependent mechanisms
  • (a) Immunoblot analysis of ERK phosphorylation in lysates from A375 following expression of GFP or COT and subsequent lentiviral shRNA-mediated MEK1 , MEK2 or MEK1 and MEK2 (MEK1 +2) depletion, relative to control shRNA (shLuc).
  • Left and right panels represent two different pairs of MEK1 and MEK2 shRNA constructs
  • Figure 23 illustrates that combinatorial MAPK pathway inhibition effectively suppresses proliferation in SKMEL28. Viability (relative to DMSO) of SKMEL28 ectopically expressing MEK1 , MEK1 DD or COT and treated with DMSO, PLX4720 (concentration indicated), PLX4720 (1 ⁇ ) and CI-1040 (1 ⁇ ) or PLX4720 (1 ⁇ ) and AZD6244 (1 ⁇ ). Error bars represent the standard deviation between replicates.
  • Figure 24 illustrates that COT over expression is sufficient to render melanoma cancer cells with the B-RAF V600E mutation resistant to B-RAF inhibition.
  • Figure 25 illustrates the top nine ORFs scoring in the primary screen were expressed in (a) SKEL28 of (b) A375 and aGI50 is shown for 4 MAPK pathway inhibitors (PLX4720, RAF265, CI-1040, AZD-6244).
  • Figure 26 illustrates that CRKL expression modifies sensitivity to the selective B-RAF inhibitor PLX4720 in a panel of B-RAFv600E cell lines.
  • Figure 27 illustrates the M/3 ⁇ 4P3K8/COT-amplified / B-RAF V600E -mutant cancer cell line OUMS-23 shows constitutive phosphorylation of ERK/MEK across a dose range of PLX4720.
  • Figure 28 illustrates the insensitivity to MAPK pathway inhibition corresponds with MAP3K8/COT copy number gains in a subset of skin cancer cell lines.
  • the present invention relates to the development of resistance to therapeutic agents in the treatment of cancer and identification of targets that confer resistance to treatment of cancer.
  • the present invention also relates to identification of parallel drug targets for facilitating an effective long-term treatment strategy and to identifying patients that would benefit from such treatment.
  • the present invention relates to kinases and in particular to MAP kinase pathway components.
  • the mitogen- activated protein kinase (MAPK) cascade is a critical intracellular signaling pathway that regulates signal transduction in response to diverse extracellular stimuli, including growth factors, cytokines, and proto-oncogenes.
  • Activation of this pathway results in transcription factor activation and alterations in gene expression, which ultimately lead to changes in cellular functions including cell proliferation, cell cycle regulation, cell survival, angiogenesis and cell migration.
  • Classical MAPK signaling is initiated by receptor tyrosine kinases at the cell surface, however many other cell surface molecules are capable of activating the MAPK cascade, including integrins, heterotrimeric G-proteins, and cytokine receptors.
  • Ligand binding to a cell surface receptor typically results in phosphorylation of the receptor.
  • the adaptor protein Grb2 associates with the phosphorylated intracellular domain of the activated receptor, and this association recruits guanine nucleotide exchange factors including SOS-I and CDC25 to the cell membrane. These guanine nucleotide exchange factors interact with and activate the GTPase Ras. Common Ras isoforms include K-Ras, N-Ras, H-Ras and others.
  • Raf serine/threonine kinase
  • A-Raf, B-Raf or Raf-1 serine/threonine kinase
  • Raf is recruited to the cell membrane through interaction with Ras.
  • Raf is then phosphorylated.
  • Raf directly activates MEKI and MEK2 by phosphorylation of two serine residues at positions 217 and 221 .
  • MEKI and MEK2 phosphorylate tyrosine (Tyr-185) and threonine (Thr-183) residues in serine/threonine kinases Erkl and Erk2, resulting in Erk activation.
  • Erk Activated Erk regulates many targets in the cytosol and also translocates to the nucleus, where it phosphorylates a number of transcription factors regulating gene expression.
  • Erk kinase has numerous targets, including Elk-I, c-Etsl, c-Ets2, p90RSKI, MNKI, MNK2, MSKI, MSK2 and TOB. While the foregoing pathway is a classical representation of MAPK signaling, there is considerable cross talk between the MAPK pathway and other signaling cascades.
  • Ras Altered expression of Ras is common in many cancers, and activating mutations in Ras have also been identified. Such mutations are found in up to 30% of all cancers, and are especially common in pancreatic (90%) and colon (50%) carcinomas. In addition, activating Raf mutations have been identified in melanoma and ovarian cancer. The most common mutation, BRAF V600E , results in constitutive activation of the downstream MAP kinase pathway and is required for melanoma cell proliferation, soft agar growth, and tumor xenograft formation. Based on the defined role of MAPK over-activation in human cancers, targeting components of the MAPK pathway with specific inhibitors is a promising approach to cancer therapy. However, patients may have innate resistance or acquire resistance to these promising therapies. Identification of target kinases, diagnostic and/or prognostic markers and treatment therapies for these patients with innate or acquired resistance are described below.
  • the present invention relates to methods of identifying targets capable of driving resistance to clinically efficacious therapies using a high throughput screening assay.
  • the method may include an open reading frame (ORF)-based functional screen for kinases that drive resistance to therapeutic agents.
  • the method may include providing a cell line with a kinase known to have an oncogenic mutation.
  • a library of kinase ORFs may be individually expressed in the cell line so that a plurality of clones each expressing a different ORF from the library may be further evaluated.
  • Each clone may be (1 ) exposed to an inhibitor of the known kinase in the cell line and (2) monitored for growth changes based on the expression of the ORF in the cell line without the inhibitor. Any clones having a growth effect from the ORF expression alone, either positive or negative growth, are eliminated. The remaining clones each expressing a different kinase are then compared for viability between a control and a treated clone and normalized to a positive control. Increased cell viability after treatment with the inhibitor identifies ORFs that confer resistance and therefore identifies kinase targets for treatment with an additional inhibitor. In some embodiments, clones scoring above two standard deviations from the normalized mean may be target kinases indicating treatment with an additional inhibitor is beneficial to the subject.
  • FIG. 5 a schematic of a high throughput functional screening assay for kinases that drive resistance to B-RAF inhibition is shown in Figure 5.
  • a collection of -600 cloned and sequence validated ORFs were assembled, accounting for -75% of all annotated kinases (Center for Cancer Systems Biology (CCSB)/Broad Institute Kinase ORF Collection, Figs. 1 a, 1 b, Table 3).
  • This publically available collection can be rapidly transferred into a variety of expression vectors for various end-applications. Any type of expression vector known to one skilled in the art may be used to express the Kinase ORF collection.
  • a selectable, epitope-tagged, lentiviral expression vector capable of producing high titer virus and robust ORF expression in mammalian cells may be created to express the kinase collection, (pLX-BLAST-V5, Figure 6a).
  • the arrayed kinase ORF collection may be stably expressed in A375, a B-RAF V600E malignant melanoma cell line that is sensitive to the RAF kinase inhibitor PLX4720 (Figs 1 a, 1 b and 6c, Table 3).
  • Clones of ORF expressing cells treated with 1 ⁇ PLX4720 are screened for viability relative to untreated cells and normalized to an assay-specific positive control, MEK1 S218 2220 (MEK1 DD ) (Table 4). ORFs that affected baseline viability or proliferation are removed from the analysis.
  • the gene encoding the target kinase may be MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL(CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3).
  • the gene encoding the target kinase may be a MAPK pathway activator.
  • the gene encoding the target kinase may be a MAP3 kinase that directly phosphorylates and activates MEK.
  • the gene encoding the target kinase may encode an adapter protein that is amplified and phosphorylated in melanoma.
  • the ORF collection may be stably expressed in a cell line having a different mutation in B-RAF, for example, another mutation at about amino acid position 600 such as V600K, V600D, and V600R. Additional B-RAF mutations include the mutations described in Davies et al. Nature, 417, 949-954, 2002, Table 1 .
  • Cell lines may be used that are sensitive to other RAF kinase inhibitors including, but not limited to, PLX4032; GDC-0879; RAF265; sorafenib; SB590855 and/or ZM 336372.
  • the ORF collection may be stably expressed in a cell line having a sensitivity to a MEK inhibitor.
  • Non-limiting examples of MEK inhibitors include, AZD6244; CI-1040; PD184352; PD318088, PD98059, PD334581 , RDEA1 19, 6- Methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinoline-3- carbonitrile and 4-[3-Chloro-4-(1 -methyl-1 H-imidazol-2-ylsulfanyl)-phenylamino]-6- methoxy-7-(3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrile. Additional RAF and MEK inhibitors are described below. By way of non-limiting example, exemplary RAF inhibitors are shown in Table 1 and exemplary MEK inhibitors are shown in Table 2.
  • the present invention relates to methods of detecting the presence of one or more diagnostic or prognostic markers in a sample (e.g. a biological sample from a cancer patient).
  • a sample e.g. a biological sample from a cancer patient.
  • a variety of screening methods known to one of skill in the art may be used to detect the presence of the marker in the sample including DNA, RNA and protein detection.
  • the techniques described below can be used to determine the presence or absence of a kinase target in a sample obtained from a patient.
  • the patient may have innate or acquired resistance to kinase targeted therapies, including B-RAF inhibitors or MEK inhibitors.
  • the patient may have an innate or acquired resistance to B-RAF inhibitors PLX4720 and/or PLX4032.
  • the patient may have innate or acquired resistance to MEK inhibitor AZD6244.
  • Identification of one or more kinase targets markers in a patient assists the physician in determining a treatment protocol for the patient. For example, in a patient having one or more kinase target markers, the physician may treat the patient with a combination therapy as described in more detail below.
  • the kinase target may include, but is not limited to, MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL(CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3).
  • the marker may be an increase in the gene copy number, an increase in protein expression, phosphorylation of one or more MAP kinase pathway members, a change in mRNA expression and the like, for the kinase target.
  • identification of an activated target kinase can be useful for characterizing a treatment protocol for the patient. For example, in a patient having a B-RAF V600E mutation, treatment with a RAF inhibitor alone may indicate that the patient is at relatively high risk of acquiring resistance to the treatment after a period of time. In a patient having an oncogenic mutation, identification of an activated kinase target in that patient may indicate inclusion of a second inhibitor in the treatment protocol.
  • Identification of an activated kinase target may include an analysis of a gene copy number and identification of an increase in copy number of a target kinase. For example, a copy number gain in MAP3K8 is indicative of a patient having innate resistance or developing acquired resistance, in particular if the patient also has a B- RAF V6OOE M UTATION
  • identification of an activated kinase target may include an analysis of phosphorylation of a kinase target and/or a member of the MAP kinase pathway.
  • phosphorylation of C-RAF at S338 is indicative of a patient having innate resistance or developing acquired resistance, in particular if the patient also has a B-RAF V600E mutation.
  • identification of an increase in MEK/ERK phosphorylation may be indicative of a patient having innate resistance or developing acquired resistance. Increased COT protein expression in patients having a B-RAF V600E mutation may predict resistance to RAF inhibition and MEK inhibition.
  • Identification of an activated kinase target may include an analysis of mRNA expression of a kinase target. For example, an increase in COT mRNA expression following initial treatment with a first kinase inhibitor is indicative of a patient having or developing resistance.
  • the first kinase inhibitor may be a RAF inhibitor or a MEK inhibitor.
  • the invention provides methods for treatment of a patient having cancer.
  • the methods generally comprise administration of a first inhibitor and a second inhibitor.
  • One inhibitor may be a RAF inhibitor.
  • the RAF inhibitor may be a pan-RAF inhibitor or a selective RAF inhibitor.
  • Pan-RAF inhibitors include but are not limited to RAF265, sorafenib, or SB590885.
  • the RAF inhibitor is a B-RAF inhibitor.
  • the selective RAF inhibitor is PLX4720, PLX4032, or GDC-0879-A.
  • One inhibitor may be a MEK inhibitor (see Table 2 illustrating exemplary MEK inhibitors).
  • One inhibitor may be a COT inhibitor.
  • the COT inhibitor may be a shRNA inhibitor as described below or a small molecule COT inhibitor, 4-(3-chloro-4-fluorophenylamino)-6-(pyridin-3-yl- methylamino)-3-cyano-[1 ,7]-naphthyridine (EMD;TPL2 inhibitor I; catalogue number 616373, PubChem ID: 9549300)
  • Inhibitors of the present invention inhibit one or more of the kinase targets including MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL(CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3) or other MAP kinase pathway targets.
  • MAP3K8 TPL2/COT
  • RAF1 CRAF
  • CrkL CrkL
  • FGR Fg
  • a combination therapy for cancer comprising an effective amount of a RAF inhibitor and an effective amount of a MAP3K8 (TPL2/COT) inhibitor.
  • a combination therapy for cancer comprising an effective amount of a RAF inhibitor and an effective amount of a MEK inhibitor.
  • Other combination therapies include an effective amount of a RAF inhibitor and an effective amount of a second inhibitor targeting the gene, mRNA or protein encoded by one or more of the following: MAP3K8 (TPL2/COT), RAF1 (CRAF),
  • the combination therapy is suitable for treatment of a patient wherein the cancer contains B-RAF mutant cells and in particular, B-RAF V600E mutant cells.
  • the present invention further provides a combination therapy for cancer, comprising an effective amount of a RAF inhibitor and an effective amount of a MEK inhibitor, wherein the subject with the cancer contains cells with altered MAP3K8 (TPL2/COT) expression or gene copy number.
  • the MEK inhibitor is CI-1040/PD184352 or AZD6244.
  • the MEK inhibitor provided herein can be Cl- 1040, AZD6244, PD318088, PD98059, PD334581 , RDEA1 19, 6-Methoxy-7-(3- morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinoline-3-carbonitrile or 4-[3- Chloro-4-(1 -methyl-1 H-imidazol-2-ylsulfanyl)-phenylamino]-6-methoxy-7-(3-morpholin-4- yl-propoxy)-quinoline-3-carbonitrile, Roche compound RG7420, or combinations thereof. Additional MEK inhibitors known in the art may also be used.
  • the RAF inhibitor provided herein is PLX4720, PLX4032, BAY 43-9006 (Sorafenib), ZM 336372, RAF 265, AAL-881 , LBT-613, or CJS352 (NVP-AAL881 -NX (hereafter referred to as
  • AAL881 and NVP-LBT613-AG-8 are isoquinoline compounds
  • combination therapy include pan-RAF inhibitors, inhibitors of B-RAF, inhibitors of A- RAF, and inhibitors of RAF-1 .
  • RAF inhibitors useful for combination therapy include PLX4720, PLX4032, BAY 43-9006 (Sorafenib), ZM 336372, RAF 265, AAL-881 , LBT-613, and CJS352.
  • Exemplary RAF inhibitors further include the compounds set forth in PCT Publication No. WO/2008/028141 , the entire contents of which are incorporated herein by reference.
  • Exemplary RAF inhibitors additionally include the quinazolinone derivatives described in PCT
  • Administration of the combination includes administration of the combination in a single formulation or unit dosage form, administration of the individual agents of the combination concurrently but separately, or administration of the individual agents of the combination sequentially by any suitable route.
  • the dosage of the individual agents of the combination may require more frequent administration of one of the agents as compared to the other agent in the combination. Therefore, to permit appropriate dosing, packaged pharmaceutical products may contain one or more dosage forms that contain the combination of agents, and one or more dosage forms that contain one of the combinations of agents, but not the other agent(s) of the combination.
  • Agents may contain one or more asymmetric elements such as stereogenic centers or stereogenic axes, e.g., asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms.
  • asymmetric elements such as stereogenic centers or stereogenic axes, e.g., asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms.
  • These compounds can be, for example, racemates or optically active forms.
  • these compounds with two or more asymmetric elements these compounds can additionally be mixtures of diastereomers.
  • compounds having asymmetric centers it should be understood that all of the optical isomers and mixtures thereof are encompassed.
  • compounds with carbon- carbon double bonds may occur in Z- and E-forms; all isomeric forms of the compounds are included in the present invention.
  • the single enantiomers can be obtained by asymmetric synthesis, synthesis from optically pure precursors, or by resolution of the racemates. Resolution of the
  • racemates can also be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example a chiral HPLC column.
  • reference to compounds useful in the combination therapy of the invention includes both the free base of the compounds, and all pharmaceutically acceptable salts of the compounds.
  • a preferred salt is the hydrochloride salt.
  • pharmaceutically acceptable salts includes derivatives of the disclosed compounds, wherein the parent compound is modified by making non-toxic acid or base addition salts thereof, and further refers to pharmaceutically acceptable solvates, including hydrates, of such compounds and such salts.
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid addition salts of basic residues such as amines; alkali or organic addition salts of acidic residues such as carboxylic acids; and the like, and combinations comprising one or more of the foregoing salts.
  • the pharmaceutically acceptable salts include non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
  • non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric; other acceptable inorganic salts include metal salts such as sodium salt, potassium salt, and cesium salt; and alkaline earth metal salts, such as calcium salt and magnesium salt; and combinations comprising one or more of the foregoing salts.
  • inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric
  • other acceptable inorganic salts include metal salts such as sodium salt, potassium salt, and cesium salt
  • alkaline earth metal salts such as calcium salt and magnesium salt
  • organic salts include salts prepared from organic acids such as acetic, trifluoroacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,
  • organic acids such as acetic, trifluoroacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic
  • n 0-4; organic amine salts such as triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, ⁇ , ⁇ '-dibenzylethylenediamine salt; and amino acid salts such as arginate, asparginate, and glutamate, and combinations comprising one or more of the foregoing salts.
  • organic amine salts such as triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, ⁇ , ⁇ '-dibenzylethylenediamine salt
  • amino acid salts such as arginate, asparginate, and glutamate, and combinations comprising one or more of the foregoing salts.
  • An "effective amount" of a combination of agents e.g., MEK and RAF inhibitors, or RAF and COT inhibitors, or RAF and an inhibitor targeting MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL(CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3)
  • RAF1 CRAF
  • FGR Fgr
  • PRKCE PRKCE
  • PRKCH Prkch
  • ERBB2 ErbB2
  • AXL Axl
  • PAK3 PAK3
  • oral dosage form is meant to include a unit dosage form prescribed or intended for oral administration.
  • An oral dosage form may or may not comprise a plurality of subunits such as, for example, microcapsules or microtablets, packaged for administration in a single dose.
  • the pharmaceutical products can be released in various forms.
  • "Releasable form” is meant to include instant release, immediate-release, controlled-release, and sustained-release forms.
  • “Instant-release” is meant to include a dosage form designed to ensure rapid dissolution of the active agent by modifying the normal crystal form of the active agent to obtain a more rapid dissolution.
  • immediate-release is meant to include a conventional or non-modified release form in which greater than or equal to about 50% or more preferably about 75% of the active agents is released within two hours of administration, preferably within one hour of administration.
  • sustained-release or extended-release includes the release of active agents at such a rate that blood (e.g., plasma) levels are maintained within a therapeutic range but below toxic levels for at least about 8 hours, preferably at least about 12 hours, more preferably about 24 hours after administration at steady-state.
  • blood e.g., plasma
  • steady-state means that a plasma level for a given active agent or
  • treat is used herein to mean to relieve, reduce or alleviate at least one symptom of a disease in a subject.
  • treatment can be diminishment of one or several symptoms of a disorder or complete eradication of a disorder, such as cancer.
  • the term “treat” also denote to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease.
  • protect is used herein to mean prevent delay or treat, or all, as appropriate, development or continuance or aggravation of a disease in a subject.
  • the disease is associated with a cancer.
  • subject or “patient” is intended to include animals, which are capable of suffering from or afflicted with a cancer or any disorder involving, directly or indirectly, a cancer.
  • subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals.
  • the subject is a human, e.g., a human suffering from, at risk of suffering from, or potentially capable of suffering from cancers.
  • the term “about” or “approximately” usually means within 20%, more preferably within 10%, and most preferably still within 5% of a given value or range. Alternatively, especially in biological systems, the term “about” means within about a log (i.e., an order of magnitude) preferably within a factor of two of a given value.
  • the instant invention provides a drug combination useful for treating, preventing, arresting, delaying the onset of and/or reducing the risk of developing, or reversing at least one symptom of cancer, in a subject comprising administering to the subject a combination therapy, comprising an effective amount of a RAF inhibitor and an effective amount of a MAP3K8 (TPL2/COT) inhibitor, or an effective amount of a RAF inhibitor and an effective amount of MEK inhibitor or an effective amount of a RAF inhibitor and an effective amount of a second inhibitor targeting MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL(CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3).
  • a combination therapy comprising an effective amount of a RAF inhibitor and an effective amount of a MAP3K8 (TPL2/COT) inhibitor, or an
  • these inhibitors are administered at therapeutically effective dosages which, when combined, provide a beneficial effect.
  • the administration may be simultaneous or sequential.
  • cancer is used herein to mean a broad spectrum of tumors, including all solid tumors and hematological malignancies.
  • tumors include but are not limited to leukemias, lymphomas, myelomas, carcinomas, metastatic carcinomas, sarcomas, adenomas, nervous system cancers and geritourinary cancers.
  • the foregoing methods are useful in treating adult and pediatric acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, anal cancer, cancer of the appendix, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma, fibrous histiocytoma, brain cancer, brain stem glioma, cerebellar astrocytoma, malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, hypothalamic glioma, breast cancer, male breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoid tumor, carcinoma of unknown origin, central nervous system lymphoma, cerebellar astrocytoma, malignant glioma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic lymphocytic
  • hypopharyngeal cancer hypothalamic and visual pathway glioma, intraocular
  • melanoma islet cell tumors, Kaposi sarcoma, kidney cancer, renal cell cancer, laryngeal cancer, lip and oral cavity cancer, small cell lung cancer, non-small cell lung cancer, primary central nervous system lymphoma, Waldenstrom macroglobulinema, malignant fibrous histiocytoma, medulloblastoma, melanoma, Merkel cell carcinoma, malignant mesothelioma, squamous neck cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndromes, myeloproliferative disorders, chronic myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblast
  • neuroectodermal tumors pituitary cancer, plasma cell neoplasms, pleuropulmonary blastoma, prostate cancer, rectal cancer, rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, squamous neck cancer, supratentorial primitive neuroectodermal tumors, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer, trophoblastic tumors, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms tumor.
  • the cancer may be associated with a mutation in the B-RAF gene.
  • These cancers include melanoma, breast cancer, colorectal cancers, glioma, lung cancer, ovarian cancer, sarcoma and thyroid cancer.
  • the therapeutic combination provided herein is effective for the treatment of moderate to severe cancer in a subject.
  • Dosages [0080] The optimal dose of the combination of agents for treatment of cancer can be determined empirically for each subject using known methods and will depend upon a variety of factors, including the activity of the agents; the age, body weight, general health, gender and diet of the subject; the time and route of administration; and other medications the subject is taking. Optimal dosages may be established using routine testing and procedures that are well known in the art.
  • the amount of combination of agents that may be combined with the carrier materials to produce a single dosage form will vary depending upon the individual treated and the particular mode of administration.
  • the unit dosage forms containing the combination of agents as described herein will contain the amounts of each agent of the combination that are typically administered when the agents are administered alone.
  • a physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the
  • a suitable daily dose of a compound of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above and is readily determined by one having skill in the art.
  • therapeutically effective doses of the compounds of this invention for a patient when used for the indicated analgesic effects, will range from about 0.0001 to about 1000 mg per kilogram of body weight per day, more preferably from about 0.01 to about 50 mg per kg per day.
  • the effective daily dose of the active compound may be any suitable daily dose of the active compound.
  • compositions comprising a combination of agents for the treatment of cancer, e.g., melanoma.
  • the pharmaceutical formulations may additionally comprise a carrier or excipient, stabilizer, flavoring agent, and/or coloring agent.
  • compositions comprising combination of agents which can be, for example, a combination of two types of agents: (1 ) a RAF inhibitor and/or pharmacologically active metabolites, salts, solvates and racemates of the inhibitor and (2) a MAP3K8 (TPL2/COT) inhibitor and/or pharmacologically active metabolites, salts, solvates and racemates of the COT inhibitor.
  • agents which can be, for example, a combination of two types of agents: (1 ) a RAF inhibitor and/or pharmacologically active metabolites, salts, solvates and racemates of the inhibitor and (2) a MAP3K8 (TPL2/COT) inhibitor and/or pharmacologically active metabolites, salts, solvates and racemates of the COT inhibitor.
  • the combination of agents may be provided for a subject comprising BRAF mutant cells or comprising cells over expressing MAP3K8 (TPL2/COT) and include: (1 ) a RAF inhibitor and/or pharmacologically active metabolites, salts, solvates and racemates of the inhibitor and (2) a MEK inhibitor and/or pharmacologically active metabolites, salts, solvates and racemates of the MEK inhibitor.
  • the combination of agents may be administered using a variety of routes of administration known to those skilled in the art.
  • the combination of agents may be administered to humans and other animals orally, parenterally, sublingually, by aerosol ization or inhalation spray, rectally, intracisternally, intravaginally,
  • Topical administration may also involve the use of transdermal administration such as transdermal patches or ionophoresis devices.
  • parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.
  • compositions for use in the present invention can be in the form of sterile, non-pyrogenic liquid solutions or suspensions, coated capsules, suppositories, lyophilized powders, transdermal patches or other forms known in the art.
  • injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
  • the sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1 ,3 propanediol or 1 ,3 butanediol.
  • the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P. and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil may be employed including synthetic mono or di glycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • the injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
  • delayed absorption of a parenterally administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle.
  • injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations may also be prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissues.
  • compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • suitable non irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
  • the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example,
  • carboxymethylcellulose alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia
  • humectants such as glycerol
  • disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium
  • the dosage form may also comprise buffering agents.
  • compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.
  • the active compounds can also be in micro-encapsulated form with one or more excipients as noted above.
  • the solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art.
  • the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch.
  • Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose.
  • the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner.
  • buffering agents include polymeric substances and waxes.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs.
  • the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, EtOAc, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3 butylene glycol,
  • the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
  • Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches.
  • the active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required.
  • Ophthalmic formulations, ear drops, and the like are also contemplated as being within the scope of this invention.
  • compositions of the invention may also be formulated for delivery as a liquid aerosol or inhalable dry powder. Liquid aerosol formulations may be nebulized predominantly into particle sizes that can be delivered to the terminal and respiratory bronchioles.
  • Aerosolized formulations of the invention may be delivered using an aerosol forming device, such as a jet, vibrating porous plate or ultrasonic nebulizer, preferably selected to allow the formation of an aerosol particles having with a mass medium average diameter predominantly between 1 to 5 m. Further, the formulation preferably has balanced osmolarity ionic strength and chloride concentration, and the smallest aerosolizable volume able to deliver effective dose of the compounds of the invention to the site of the infection. Additionally, the aerosolized formulation preferably does not impair negatively the functionality of the airways and does not cause undesirable side effects.
  • an aerosol forming device such as a jet, vibrating porous plate or ultrasonic nebulizer
  • Aerosol ization devices suitable for administration of aerosol formulations of the invention include, for example, jet, vibrating porous plate, ultrasonic nebulizers and energized dry powder inhalers, that are able to nebulize the formulation of the invention into aerosol particle size predominantly in the size range from 1 5 m.
  • a jet nebulizer works by air pressure to break a liquid solution into aerosol droplets.
  • Vibrating porous plate nebulizers work by using a sonic vacuum produced by a rapidly vibrating porous plate to extrude a solvent droplet through a porous plate.
  • An ultrasonic nebulizer works by a piezoelectric crystal that shears a liquid into small aerosol droplets.
  • a variety of suitable devices are available, including, for example, AERONEB and AERODOSE vibrating porous plate nebulizers (AeroGen, Inc., Sunnyvale, California), SIDESTREAM nebulizers (Medic Aid Ltd., West Wales, England), PARI LC and PARI LC STAR jet nebulizers (Pari Respiratory Equipment, Inc., Richmond, Virginia), and AEROSONIC (DeVilbiss Medizinische discipline (Deutschland) GmbH, Heiden, Germany) and
  • Compounds of the invention may also be formulated for use as topical powders and sprays that can contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances.
  • Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
  • Transdermal patches have the added advantage of providing controlled delivery of a compound to the body.
  • dosage forms can be made by dissolving or dispensing the compound in the proper medium.
  • Absorption enhancers can also be used to increase the flux of the compound across the skin.
  • the rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel.
  • the compounds of the present invention can also be
  • liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono or multi lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used.
  • the present compositions in liposome form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients, and the like.
  • the preferred lipids are the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott (ed.), "Methods in Cell Biology,” Volume XIV, Academic Press, New York, 1976, p. 33 et seq.
  • Example 1 An ORF-based functional screen identifies specific kinases as drivers of resistance to B-RAF inhibition.
  • ORF expressing cells treated with 1 ⁇ PLX4720 were screened for viability relative to untreated cells and normalized to an assay-specific positive control, MEK1 S218 222D (MEK1 DD ) (Emery, C. M. et al. Proc. Natl Acad. Sci. USA 106, 2041 1-20416 (2009).) (Table 4 and summarized in Fig. 5).
  • MEK1 S218 222D MEK1 DD
  • Fig. 5 Nine ORFs conferred resistance at levels exceeding two standard deviations from the mean (Fig. 1 b and Table 4) and were selected for follow-up analysis (Fig. 7).
  • Three of nine candidate ORFs were receptor tyrosine kinases, underscoring the potential of this class of kinases to engage resistance pathways.
  • tyrosine kinases such as BCR-ABL (Birge, R.B. et al., Cell Commun Signal 7, 13 (2009)), but lacks intrinsic kinase activity.
  • COT and C-RAF reduced sensitivity to PLX4720 in multiple B-RAF V600E cell lines (Fig. 1c) confirming the ability of these kinases to mediate resistance to RAF inhibition.
  • a secondary screen in A375 and SKMEL28 prioritizes the top 9 candidate ORFs across a multipoint PLX4720 concentration scale (Fig. 1 d).
  • MAP3Ks Ser/Thr MAP kinase kinase kinases
  • C-RAF is a MAP3K in the canonical MAPK cascade (McKay, M.M. and Morrison, D.K. Oncogene 26, 31 13-3121 (2007)) that was previously implicated in resistance associated with stepwise selection in vitro using a pan-RAF inhibitor
  • COT the protein product of the human MAP3K8 gene
  • MAP3K the protein product of the human MAP3K8 gene
  • MAP3K downstream of NFKB signaling in inflammatory cells
  • Example 2 Resistance to B-RAF inhibition via MAPK pathway activation.
  • Example 3 C-RAF activation and heterodimerization with B-RAF.
  • C-RAF activation and heterodimerization with B-RAF constitute critical components of the cellular response to B-RAF inhibition.
  • endogenous C-RAF B-RAF heterodimers were measurable and inducible following treatment with PLX4720 (Fig. 13).
  • C-RAF was phosphorylated on S338 (Fig. 13) and its PLX4720 resistance phenotype was associated with sustained MEK/ERK activation (Figs. 2a and 13).
  • C-RAF(W22) a high-activity C-RAF truncation mutant
  • Fig. 14 ectopic expression of a high-activity C-RAF truncation mutant
  • Fig. 14 ectopic expression of a high-activity C-RAF truncation mutant
  • Fig. 14 ectopic expression of a high-activity C-RAF truncation mutant
  • Fig. 14 ectopic expression of a high-activity C-RAF truncation mutant
  • Fig. 14 ectopic expression of a high-activity C-RAF truncation mutant
  • Example 5 COT expression predicts resistance to B-RAF inhibition in cancer cell lines.
  • a panel of melanoma short-term cultures was also screened for COT protein expression.
  • One of these lines expressed COT M307, a short-term culture derived from a B-RAF V600E tumor that developed resistance to allosteric MEK inhibition following initial disease stabilization (Fig. 3c). All three cell lines were refractory to PLX4720 treatment, exhibiting GI 5 0 values in the range of 8-10 ⁇ (Fig. 3d) and showing sustained ERK phosphorylation in the context of B-RAF inhibition (Figs. 3e, 3f).
  • OUMS-23 and RPMI-7951 are MAPK pathway inhibitor-na ' fve cell lines; thus, these results demonstrate that COT confers de novo resistance to RAF inhibition (a
  • Example 6 COT expression in patients treated with a RAF inhibitor.
  • COT expression in the context of resistance to the clinical RAF inhibitor PLX4032 was examined by obtaining biopsy material from 3 patients with metastatic, B-RAF V600E melanoma. Each case consisted of frozen, lesion-matched biopsy material obtained prior to and during treatment ("pre-treatment” and “on-treatment”; Fig. 3g, Table 6); additionally, one sample contained two independent biopsy specimens from the same relapsing tumor site ("post-relapse”; Fig. 3g). Consistent with the
  • Example 7 COT regulation of MEK/ERK phosphorylation.
  • RPMI-7951 viability (Fig. 20) and decreased ERK phosphorylation (Fig. 3h); thus, targeting COT kinase activity suppresses MEK/ERK phosphorylation in cancer cells with COT overexpression or amplification. Additionally, the targeting COT kinase activity in the presence of a B-RAF inhibitor (PLX4720) suppresses MEK/IRK phosphorylation (Fig. 3h).
  • a small molecule COT kinase inhibitor (Wyeth, Abbot compound ID 9549300) (George, D. et al., Bioorg. Med. Chem. Lett. 18, 4952-4955 (2008); Hirata, K. et al., Biol.
  • Example 8 COT-expressing B-RAF V600E cell lines exhibit resistance to allosteric MEK inhibitors.
  • COT-expressing cancer cells remain sensitive to MAPK pathway inhibition at a target downstream of COT or RAF was analyzed.
  • the OUMS-23 and RPMI-7951 cell lines were queried for sensitivity to the MEK1/2 inhibitor CI-1040. Both cell lines were refractory to MEK inhibition (Fig. 4a) and displayed sustained ERK phosphorylation even at 1 ⁇ CI-1040 (Fig. 4b).
  • Ectopic COT expression in A375 and SKMEL28 cells also conferred decreased sensitivity to the MEK inhibitors CI-1040 and AZD6244, suggesting that COT expression alone was sufficient to induce this
  • Example 9 Combinatorial MAPK pathway inhibition to suppress cell proliferation.
  • kinases ORFs were assembled from multiple sources; 337 kinases were isolated as single clones from the ORFeome 5.1 collection (http://horfdb.dfci.harvard.edu), 183 kinases were cloned from normal human tissue RNA (Ambion) by reverse transcription and subsequent PCR amplification to add Gateway sequences (Invitrogen), 64 kinases were cloned from templates provided by the Harvard Institute of Proteomics (HIP), and 13 kinases were cloned into the Gateway system from templates obtained from collaborating laboratories.
  • HIP Harvard Institute of Proteomics
  • the Gateway- compatible lentiviral vector pLX-Blast-V5 was created from the pLKO.1 backbone. LR Clonase enzymatic recombination reactions were performed to introduce the 597 kinases into pLX-Blast-V5 according to the manufacturer's protocol (Invitrogen).
  • A375 melanoma cells were plated in 384-well microtiter plates (500 cells per well). The following day, cells were spin-infected with the lentivirally-packaged kinase ORF library in the presence of 8 ug/ml polybrene. 48 hours post-infection, media was replaced with standard growth media (2 replicates), media containing 1 ⁇
  • PLX4720 (2 replicates, 2 time points) or media containing 10 ug/ml blasticidin (2 replicates). After four days and 6 days, cell growth was assayed using Cell Titer-Glo (Promega) according to manufacturer instructions. The entire experiment was performed twice.
  • MEK1 DD normalized differential proliferation for each individual ORF was averaged across two duplicate experiments, with two time points for each experiment (day 4 and day 6). A z-score was then generated, as described above for average MEK1 DD normalized differential proliferation. ORFs with a z-score of >2 were considered hits and were followed up in the secondary screen.
  • ORFs were expressed from pLX-Blast-V5 (lentiviral) or pWZL-Blast, pBABE-Puro or pBABE-zeocin (retroviral) expression plasmids.
  • lentiviral transduction 293T cells were transfected with 1 ⁇ g of pLX-Blast-V5-ORF or pLKO.
  • Mammalian cells were infected at a 1 : 10-1 :20 dilution of virus in 6-well plates in the presence of 5 ⁇ g/ml polybrene and centrifuged at 2250 RPM for 1 h at 37°C. Twenty-four hours after infection blasticidin (pLX-Blast-V5, 10 ⁇ g/ml) or puro (pLKO.1 , 0.75 ⁇ g/ml) was added and cells were selected for 48 hrs. For retrovirus production, 293T were transfected with 1 ⁇ g of retroviral plasmid-ORF, 1 ⁇ g pCL-AMPHO and 100 ng VSV-G, as described above.
  • A375 (1 .5 x 10 3 ) and SKMEL28 cells (3 x 10 3 ) were seeded in 96-well plates for 18 h.
  • ORF-expressing lentivirus was added at a 1 : 10 dilution in the presence of 8 ⁇ g/ml polybrene, and centrifuged at 2250 RPM and 37° C for 1 h. Following centrifugation, virus-containing media was changed to normal growth media and allowed to incubate for 18 h.
  • DMSO (1 : 1000) or 10x PLX4720 (in DMSO) was added to a final concentration of 100, 10, 1 , 0.1 , 0.01 , 0.001 , 0.0001 or 0.00001 ⁇ .
  • Cell viability was assayed using WST-1 (Roche), per manufacturer recommendation, 4 days after the addition of PLX4720.
  • A375, SKMEL28, SKMEL30, COLO-679, WM451 lu, SKMEL5, Malme 3M, SKMEL30, WM3627, WM1976, WM3163, WM3130, WM3629, WM3453, WM3682 and WM3702 were all grown in RPMI (Cellgro), 10% FBS and 1 % penicillin/streptomycin.
  • M307 was grown in RPMI (Cellgro), 10% FBS and 1 % penicillin/streptomycin supplemented with 1 mM sodium pyruvate. 293T and
  • OUMS-23 were grown in DMEM (Cellgro), 10% FBS and 1 % penicillin/streptomycin.
  • RPMI-7951 cells ATCC were grown in MEM (Cellgro), 10% FBS and 1 %
  • CI-1040 (PubChem ID: 6918454) was purchased from Shanghai Lechen International Trading Co., AZD6244
  • RAF265 (PubChem ID: 1 1656518) was a generous gift from Novartis Pharma AG. Unless otherwise indicated, all drug treatments were for 16 h. Activated alleles of NRAS and KRAS have been previously described.
  • Protein was transferred to PVDF membranes and probed with primary antibodies recognizing pERK1/2 (T202/Y204), pMEK1/2 (S217/221 ), MEK1/2, MEK1 , MEK2, C-RAF (rabbit host), pC-RAF (pS338) (Cell Signaling Technology; 1 : 1 ,000), V5-HRP (Invitrogen; (1 :5,000), COT (1 :500), B-RAF (1 :2,000), Actin (1 :1 ,000), Actin-HRP (1 :1 ,000; Santa Cruz)), C-RAF (mouse host; 1 :1 ,000; BD Transduction Labs), Vinculin (Sigma; 1 :20,000), AXL (1 :500; R&D Systems).
  • Lysates from tumor and matched normal skin were generated by mechanical homogenization of tissue in RIPA [50 mM Tris (pH 7.4), 150 mM NaCI, 1 mM EDTA, 0.1 % SDS, 1 .0% NaDOC, 1 .0% Triton X-100, 25 mM NaF,
  • Biopsied tumor material consisted of discarded and de-identified tissue that was obtained with informed consent and characterized under protocol 02-017 (paired samples, Massachusetts General Hospital) and 07-087 (unpaired sample, Dana-Farber Cancer Institute). For paired specimens, ⁇ -treatment' samples were collected 10-14 days after initiation of PLX4032 treatment (Table 6).
  • Adherent RPMI-7951 cells were infected with virus expressing shRNAs against COT or Luciferase as described above. Following selection, cells were plated (1 .5x10 5 cells/well) onto a 24-well plate in quadruplicate. Viable cells were counted via trypan blue exclusion using a VI-CELL Cell Viability Analyzer, per manufacturer's specifications. Quadruplicate cell counts were averaged and normalized relative to that of the control shRNA.
  • CCLE Cancer Cell Line Encyclopedia
  • Oligonucleotide microarray analysis was carried out using the GeneChip Human Genome U133 Plus 2.0 Affymetrix expression array (Affymetrix). Samples were converted to labeled, fragmented, cRNA following the Affymetrix protocol for use on the expression microarray.
  • RS/TK Receptor Serine/Threonine Kinase
  • RTK Receptor Tyrosine Kinase
  • NRS/TK Non-Receptor Serine/Threonine Kinase
  • NRTK Non-Receptor Receptor Serine/Threonine Kinase
  • Table 4 Ranking of average differential proliferation (1 ⁇ PLX4720/control) for 597 kinase-
  • PAK3 0.493 2.95 9 PAK3 0.9041 0.9041 9

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Abstract

A method of identifying a subject having cancer who is likely to benefit from treatment with a combination therapy with a RAF inhibitor and a second inhibitor is provided. A method of treating cancer in a subject in need thereof is also provided and includes administering to the subject an effective amount of a RAF inhibitor and an effective amount of a second inhibitor, wherein the second inhibitor is a MEK inhibitor, a CRAF inhibitor, a CrkL inhibitor or a TPL2/COT inhibitor. A method of identifying a kinase target that confers resistance to a first inhibitor is also provided.

Description

METHODS OF DIAGNOSING AND TREATING CANCER IN PATI ENTS HAVING OR DEVELOPING RESISTANCE TO A FI RST CANCER THERAPY
RELATED APPLICATIONS
[0001] This application claims the benefit of the filing date under 35 U.S.C.
§1 19(e) of the following Provisional U.S. Patent Application Serial Nos.: 61/312,193, filed March 9, 2010; 61/312,519, filed March 10, 2010; 61/326,021 , filed April 20, 2010; and 61/415,569, filed November 19, 2010, all of which are hereby
incorporated by reference in their entirety.
FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under federal grant numbers K08 CA1 15927 and 1 DP20D002750 awarded by National Institutes of Health. The government has certain rights in the invention.
BACKGROUND
[0003] Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. (Davies, H. et al., Nature 417, 949-954 (2002).) Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma (Hoeflich, K. P. et al., Cancer Res. 69, 3042-3051
(2009) ; McDermott, U. et al., Proc. Natl Acad. Sci. USA 104, 19936-19941 (2007); Solit, D. B. et al. BRAF mutation predicts sensitivity to MEK inhibition. Nature 439, 358-362 (2006); Wan, P. T. et al., Cell 1 16, 855-867 (2004); Wellbrock, C. et al., Cancer Res. 64, 2338-2342 (2004))— an observation that has been validated by the success of RAF or MEK inhibitors in clinical trials (Flaherty, K. T. et al., N. Engl. J. Med. 363, 809-819
(2010) ; Infante, J. R. et al., J. Clin. Oncol. 28 (suppl.), 2503 (2010); Schwartz, G. K. et al., J. Clin. Oncol. 27 (suppl.), 3513 (2009).) However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. (Engelman, J. A. et al., Science 316, 1039-1043 (2007); Gorre, M. E. et al., Science 293, 876-880 (2001 ); Heinrich, M. C. et al., J. Clin. Oncol. 24, 4764-4774 (2006); Daub, H., Specht, K. & Ullrich, A. Nature Rev. Drug Discov. 3, 1001-1010 (2004).) Accordingly, there remains a need for new methods for identification of resistance mechanisms in a manner that elucidates "druggable" targets for effective long-term treatment strategies, for new methods of identifying patients that are likely to benefit from the treatment strategies, and for methods of treating patients with the effective long-term treatment strategies.
BRI EF SUMMARY
[0004] The present invention relates to the development of resistance to therapeutic agents in the treatment of cancer and identification of targets that confer resistance to treatment of cancer. The present invention also relates to identification of parallel drug targets for facilitating an effective long-term treatment strategy and to identifying patients that would benefit from such treatment.
[0005] Accordingly, in one aspect, a method of identifying a subject having cancer who is likely to benefit from treatment with a combination therapy with a RAF inhibitor and a second inhibitor is provided. The method includes assaying a gene copy number, a mRNA or a protein level or phosphorylation of one or more kinase targets selected from the group consisting of MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL(CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3) in cancer cells obtained from the subject. The method further includes comparing the gene copy number, the mRNA or the protein level or the phoshorylation with a gene copy number, a mRNA or a protein level or phosphorylation of the target kinase in cells obtained from a subject without the cancer and correlating increased gene copy number or an alteration in mRNA expression or protein overexpression or phosphorylation of the target kinase in the cancer cells relative to the cells from the subject without the cancer with the subject having the cancer who is likely to benefit from treatment with the combination therapy.
[0006] In another aspect, a method of treating cancer in a subject in need thereof is provided. The method includes administering to the subject an effective amount of a RAF inhibitor and an effective amount of a second inhibitor, wherein the second inhibitor is a MEK inhibitor or a TPL2/COT inhibitor. [0007] In another aspect, a method of identifying a kinase target that confers resistance to a first inhibitor is provided. The method includes culturing cells having sensitivity to the first inhibitor and expressing a plurality of kinase ORF clones in the cell cultures, each cell culture expressing a different kinase ORF clone. The method further includes exposing each cell culture to the inhibitor and identifying cell cultures having greater viability than a control cell culture after exposure to the inhibitor to identify the kinase ORF clone that confers resistance to the first inhibitor.
BRIEF DESCRIPTION OF THE DRAWINGS
[0008] Figure 1 illustrates an ORF-based functional screen which identified COT and C-RAF kinases as drivers of resistance to B-RAF inhibition, (a) Schematic overview of the CCSB/Broad Institute Kinase ORF collection. Kinase classification and number of kinases per classification are noted; (b) A375 cells expressing the
CCSB/Broad Institute Kinase ORF collection were assayed for relative viability in 1 μΜ PLX4720 and normalized to constitutively active MEK1 (MEK1 DD). Nine ORFs (circles) scored 2 standard deviations (dashed line, 58.64%) from the mean of all ORFs (dashed line, 44.26%); (c) Indicated ORFs were expressed in 5 B-RAFV600E cell lines and treated with DMSO or 1 μΜ PLX4720. Viability (relative to DMSO) was quantified after 4 days. Error bars represent standard deviation between replicates (n=6); (d) Secondary screen in A375 and SKMEL28 prioritizes the top 9 candidate ORFs across a multipoint
PLX4720 concentration scale.
[0009] Figure 2 illustrates resistance to B-RAF inhibition via MAPK pathway activation, (a) Indicated ORFs were expressed in A375. Levels of phosphorylated MEK and ERK were assayed following 18 h. treatment with DMSO (-) or PLX4720
(concentration noted); (b) Proliferation of A375 expressing indicated ORFs. Error bars represent standard deviation between replicates (n=6). (c) C-RAF (S338) and ERK phosphorylation in lysates from A375 expressing indicated ORFs. (d) COT expression in lysates from immortalized primary melanocytes expressing BRAFV600E or empty vector. COT mRNA has an internal start codon (30M) resulting in two protein products of different lengths; amino acids 1 -467 or 30-467, noted with arrows, (e) COT and ERK phosphorylation in lysates from A375 expressing indicated ORFs following shRNA-mediated B-RAF depletion (shBRAF) relative to control shRNA (shLuc). (f) ERK phosphorylation in lysates from A375 expressing indicated ORFs following
shRNA-mediated C-RAF depletion (shCRAF) or control shRNA (shLuc), following 18 h. treatment with DMSO (-) or 1 μΜ PLX4720 (+).
[0010] Figure 3 illustrates COT expression predicts resistance to B-RAF inhibition in cancer cell lines, (a) MAP3K8/COT copy number; red bars: COT amplification, blue bars: non-amplified COT; (b) COT expression in B-RAFV600E cell lines and (c) short-term cultures; (d) PLX4720 GI50 in B-RAFV600E cell lines. Colors as in (a); (e) MEK and ERK phosphorylation following treatment with DMSO or PLX4720 (concentration indicated); (f) ERK phosphorylation in M307 lysates (AZD-R; AZD6244-resistant) treated with DMSO or 1 μΜ PLX4720 (PLX) or CI-1040 (CI); (g) COT mRNA expression (QRT/PCR) in patient/lesion-matched PLX4032-treated metastatic melanoma tissue samples. Pts. 1 and 3 had multiple biopsies from the same lesion. Error bars represent SEM (n=3) U; undetermined/undetectable; (h) ERK and MEK phosphorylation in RPMI-7951 following shRNA-mediated COT depletion (shCOT) versus control (shLuc) and treatment with DMSO (-) or 1 μΜ PLX4720 (+). ERK and MEK phosphorylation are quantified; (i) ERK and MEK phosphorylation in RPMI-7951 following 1 h. treatment with a small molecule COT kinase inhibitor. ERK and MEK phosphorylation are quantified, (j) PLX4720 sensitivity curves in a panel of BRAFV600E cell lines. OUMS-23 and M307 represent cell lines with COT expression/amplification and all others represent cell lines with undetectable/unaltered COT; (k) Selective expression of COT and corresponding MAPK pathway activation in a metastatic subcutaneous malignant melanoma with acquired resistance to PLX4032 (* denotes a background band, MET-MM (PLX-R); metastatic malignant melanoma, PLX4032-resistant).
[0011] Figure 4 illustrates COT-expressing B-RAFV600E cell lines exhibit resistance to allosteric MEK inhibitors, (a) CI-1040 GI50 in a panel of B-RAFV600E cell lines; (b) MEK and ERK phosphorylation in lysates from indicated cell lines treated with DMSO or CI-1040 (concentration noted); (c) Fold change (relative to MEK1 ) GI50 of A375 ectopically expressing the indicated ORFs for PLX4720, RAF265, CI-1040 and AZD6244; (d) ERK phosphorylation in A375 expressing indicated ORFs following treatment with DMSO or 1 μΜ of PLX4720, RAF265, CI-1040 or AZD6244; (e) Viability of A375 expressing the indicated ORFs and treated with DMSO, PLX4720 (concentration indicated) and PLX4720 in combination with CI-1040 or AZD6244 (all 1 μΜ). Error bars represent the standard deviation (n=6); (f) ERK phosphorylation in A375 expressing indicated ORFs following treatment with DMSO, PLX4720 (1 μΜ) or
PLX4720 in combination with CI-1040 or AZD6244 (all 1 μΜ); (g) Cell lines with aberrant MAP3K8/COT copy number/expression are insensitive to the allosteric MEK inhibitor CI-1040 or (h) AZD6244; (i) A schematic outlining the formation of MAP3K complexes in response to B-RAF inhibition in B-RAFV600E-mutant cell lines. PLX4720 positions C-RAF in a signaling-competent complex (upper right panel) that is activated by oncogenic events upstream of C-RAF (lower right panel), subsequently driving resistance. In the context of COT expression, COT/RAF-containing complexes are sufficient to activate the MAPK pathway and mediate resistance (lower left panel).
[0012] Figure 5 illustrates a schematic outline of an ORF-based functional screen for kinases that drive resistance to B-RAF inhibition. The B-RAFV600E cell line A375 was lentivirally transduced with the 597 kinases in the CCSB/Broad Institute Kinase ORF Collection. ORFs having a positive or negative effect on proliferation in control-treated A375 were identified and removed from final analysis. Resistance-promoting ORFs were identified by generating a differential viability ratio between B-RAF inhibited (PLX4720-treated) and control-treated cells. Differential viability was normalized to a constitutively active MEK1 allele, MEK1 DD; an assay specific positive control.
[0013] Figure 6 illustrates that the CCSB/Broad Institute Kinase ORF Collection is well expressed via high titer lentivirus. (a), schematic of the pLX-BLAST-V5 lentiviral expression vector used for all ORF-screens and subsequent validation, (b) GFP-tagged ORFs representing a broad size range were lentivirally expressed in Jurkat cells and the percentage of GFP-expressing cells/ORF (e.g., infected cells) quantified, demonstrating high viral titer across a range of ORF sizes, (c), expression of 96 random ORFs detected via LiCor with antibodies against the V5-epitope tag, relative to cellular DNA. Expression was detectable in 83% of the wells.
[0014] Figure 7 illustrates the expression of candidate resistance ORFs. 293T were transiently transfected with pLX-BLAST-V5-ORF (indicated) and expression detected using an anti-V5-HRP antibody. The AXL clone is 'closed' and has a stop codon preceding the V5 tag. See Figure 12 for verification of expression; (*) on dark-exposure indicate the expression of three ORFs not visible in the lighter exposure.
[0015] Figure 8 illustrates that a secondary screen prioritizes the top 9 candidate B-
RAF inhibitor resistance ORFs. The top nine ORFs scoring in the primary screen were expressed in A375 or SKMEL28 and a Gl50 from an 8-point PLX4720 concentration range.
[0016] Figure 9 illustrates the effects of ORF expression on proliferation in B- RAFV600E cell lines. Proliferation, relative to MEK1 , in (a) A375 or (b) SKMEL28 expressing indicated ORFs after 7 days of growth.
[0017] Figure 10 illustrates that ectopic expression of constitutively active MEK1 (MEK1 DD) and COT lead to increased pMEK/pERK in A375, whereas C-RAF reduces pMEK/pERK levels. Lysates from A375 ectopically expressing GFP, MEK1 , MEKDD, COT or C-RAF were analyzed via immunoblot for levels of pERK and pMEK. GFP and MEK1 (lanes 1 -3) were separated from COT/C-RAF (lanes 4-5) to prevent residual V5- MEK1 signal from overwhelming that of COT and C-RAF, which are expressed at much lower levels.
[0018] Figure 11 illustrates that the kinase activity of COT and C-RAF is required for sustained ERK phosphorylation in the context of PLX4720 treatment. Immunoblot analysis of A375 expressing ectopic (a) MEK1 , wild type COT or kinase inactive COT (COTK167R) or (b) MEK1 , wild type C-RAF or kinase inactive C-RAF (C-RAFK375M) treated with 1 μΜ PLX4720 for 18 h.
[0019] Figure 12 illustrates the effects of ORF expression on MAPK signaling in the context of the B-RAF inhibitor PLX4720. MAPK pathway activation was assessed by immunoblot analysis of pERK and pMEK in A375 expressing the indicated ORFs in the presence of PLX4720 (18 h., concentration indicated). (*) indicates the use of an antibody directed against the expressed ORF, not the V5 epitope. AXL was cloned without the V5 tag.
[0020] Figure 13 illustrates that B-RAF associates with immunoprecipitated C-RAF in A375 following 18 hr. treatment with 1 μΜ PLX4720 (+) or DMSO (-), (a). WCE; whole cell extract controls. Ectopically expressed C-RAF constitutively associates with B-RAF and is phosphorylated at S338, consistent with membrane localization and activation. MEK1 , MEKDD and COT-expressing A375 show no evidence of C-RAF activation, (b).
[0021] Figure 14 illustrates that Retroviral expression of a wild-type C-RAF or a high-activity truncation mutant of C-RAF (C-RAF(22W)) renders A375 resistant to the B- inhibitor PLX4720 (a) and leads to sustained pERK levels in the context of PLX4720 treatment (1 μΜ, 18 h.), (b). C-RAF expression levels achieved with retroviruses are significantly lower than with lentiviral-based systems, resulting in a lower GI5o than that achieved with lentiviral C-RAF.
[0022] Figure 15 illustrates the effects of B-RAFV600E on COT mRNA (a)
Quantitative RT/PCR of COT mRNA expression relative to GAPDH mRNA expression in transformed primary melanocytes expressing wild-type B-RAF (vector) or B-RAFV600E. COT expression was normalized to that of vector-expressing primary melanocytes. (**) Significant, p 0.05 (Student's two-tailed, paired T-Test). Endogenous COT mRNA is undetectable in PLX4720-sensitive A375 and ectopically expressed COT mRNA levels are unaffected by 1 μΜ PLX4720 treatment. A375 expressing GFP or COT were treated for 18 h. with 1 μΜ PLX4720. Reverse-transcribed mRNA was analyzed for GAPDH- normalized COT expression, relative to GFP-expressing, DMSO treated, A375. (*) Not significant, p > 0.05 (Student's two-tailed, paired T-Test). Error bars represent SEM.
[0023] Figure 16 illustrates that B- and C-RAF protein levels are not required for COT-mediated ERK phosphorylation. A375 expressing ectopic MEK1 (control) or COT were sequentially infected with lentivirus expressing shRNAs targeting B-RAF, C-RAF or control shRNA (shLuc) and assayed for expression of the indicated proteins in the presence (+) or absence (-) of 1 μΜ PLX4720, 18 h.
[0024] Figure 17 illustrates SNP analysis of 752 cell lines reveals copy number alterations in MAP3K8/COT. Of the 752 cell lines hat had undergone copy number analysis, 534 had also undergone mutation profiling. Thirty-eight (7.1 %) of mutation- profiled cells harbor the B-RAFV600E mutation. Two cell lines (OUMS-23, RPMI-7951 , indicated) harbor the B-RAFV600E mutation along with copy number gain in
MAP3K8/COT.
[0025] Figure 18 illustrates MAP3K8/COT alterations in the cancer cell line OUMS- 23. (a) RMA signal of a MAP3K8 /COT probe (noted) from mRNA microarray analysis. OUMS-23 is one of the top 2% (of 765 cell lines) expressing COT mRNA. (b) COT mRNA expression in a panel of B-RAFV600E-mutant cell lines, (c) Endogenous COT protein expression in OUMS-23 relative to ectopically expressed COT in A375 and SKMEL28 cell lines, as determined via immunoblot analysis of the indicated cells.
[0026] Figure 19 illustrates that COT mRNA and protein are expressed in B RAF- inhibitor resistant cell lines and tissue, (a) RT/PCR analysis of GAPDH normalized COT mRNA expression in a panel of cell lines, short term cultures and tissue from relapsed, PLX4032-treated, malignant melanoma (MM-R). Corresponding protein expression for cell lines and short term cultures are shown in Figures 3b and 3c, respectively, (b) Western blot analysis of lysates from primary melanocytes (1 ° Mel(B-RAF WT)), patient matched normal skin (Skin) and metastatic malignant melanoma (MM-R; COT mRNA shown in panel a), A375 cells and primary melanocytes expressing B-RAFV600E (1 ° Mel (B-RAFV600E)).
[0027] Figure 20 illustrates that depletion of COT affects viability in the COT amplified cell line RPMI-7951 . (a) Quantification of RPMI-7951 viability following lentiviral shRNA-mediated COT depletion (shCOT), relative to control shRNA (shLuc). Error bars represent standard deviation between replicates, (b) Immunoblot analysis showing relative COT protein expression in shLuc and shCOT-expressing RPMI-7951 .
[0028] Figure 21 illustrates effects of ORF expression on the GI5o of a panel of MAPK pathway inhibitors in SKMEL28. The half-maximal growth-inhibitory
concentration (GI5o) of SKMEL28 ectopically expressing MEK1 , MEK1 DD or COT was determined for the RAF inhibitors PLX4720 and RAF265 and the MEK1 /2 inhibitors Cl- 1040 and AZD6244. The change in Gl50 for MEK1 DD and COT (relative to MEK1 ) was determined for each compound.
[0029] Figure 22 illustrates that COT can activate ERK via MEK-independent and MEK-dependent mechanisms, (a) Immunoblot analysis of ERK phosphorylation in lysates from A375 following expression of GFP or COT and subsequent lentiviral shRNA-mediated MEK1 , MEK2 or MEK1 and MEK2 (MEK1 +2) depletion, relative to control shRNA (shLuc). Left and right panels represent two different pairs of MEK1 and MEK2 shRNA constructs, (b) Immunoblot blot analysis of recombinant, inactive ERK phosphorylation (Thr202/Tyr204) by recombinant COT in an in vitro kinase assay. [0030] Figure 23 illustrates that combinatorial MAPK pathway inhibition effectively suppresses proliferation in SKMEL28. Viability (relative to DMSO) of SKMEL28 ectopically expressing MEK1 , MEK1 DD or COT and treated with DMSO, PLX4720 (concentration indicated), PLX4720 (1 μΜ) and CI-1040 (1 μΜ) or PLX4720 (1 μΜ) and AZD6244 (1 μΜ). Error bars represent the standard deviation between replicates.
[0031] Figure 24 illustrates that COT over expression is sufficient to render melanoma cancer cells with the B-RAFV600E mutation resistant to B-RAF inhibition.
[0032] Figure 25 illustrates the top nine ORFs scoring in the primary screen were expressed in (a) SKEL28 of (b) A375 and aGI50 is shown for 4 MAPK pathway inhibitors (PLX4720, RAF265, CI-1040, AZD-6244).
[0033] Figure 26 illustrates that CRKL expression modifies sensitivity to the selective B-RAF inhibitor PLX4720 in a panel of B-RAFv600E cell lines.
[0034] Figure 27 illustrates the M/¾P3K8/COT-amplified / B-RAFV600E-mutant cancer cell line OUMS-23 shows constitutive phosphorylation of ERK/MEK across a dose range of PLX4720.
[0035] Figure 28 illustrates the insensitivity to MAPK pathway inhibition corresponds with MAP3K8/COT copy number gains in a subset of skin cancer cell lines. A panel of 20 B-RAFV600E-mutant cell lines and their sensitivity to (a) the B-RAF inhibitor PLX4720 and (b) the MEK inhibitor AZD6244 is shown.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The present invention relates to the development of resistance to therapeutic agents in the treatment of cancer and identification of targets that confer resistance to treatment of cancer. The present invention also relates to identification of parallel drug targets for facilitating an effective long-term treatment strategy and to identifying patients that would benefit from such treatment. In some embodiments, the present invention relates to kinases and in particular to MAP kinase pathway components.
[0037] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, immunology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See e.g., Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, (Current Edition); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel et al. eds., (Current Edition)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (Current Edition) ANTIBODIES, A LABORATORY MANUAL and ANIMAL CELL CULTURE (R. I. Freshney, ed. (1987)). DNA Cloning: A Practical Approach, vol. I & II (D. Glover, ed.); Oligonucleotide Synthesis (N. Gait, ed., Current Edition); Nucleic Acid Hybridization (B. Hames & S. Higgins, eds., Current Edition); Transcription and Translation (B. Hames & S. Higgins, eds., Current Edition); Fundamental Virology, 2nd Edition, vol. I & II (B. N. Fields and D. M. Knipe, eds.)
[0038] The mitogen- activated protein kinase (MAPK) cascade is a critical intracellular signaling pathway that regulates signal transduction in response to diverse extracellular stimuli, including growth factors, cytokines, and proto-oncogenes.
Activation of this pathway results in transcription factor activation and alterations in gene expression, which ultimately lead to changes in cellular functions including cell proliferation, cell cycle regulation, cell survival, angiogenesis and cell migration.
Classical MAPK signaling is initiated by receptor tyrosine kinases at the cell surface, however many other cell surface molecules are capable of activating the MAPK cascade, including integrins, heterotrimeric G-proteins, and cytokine receptors.
[0039] Ligand binding to a cell surface receptor, e.g., a receptor tyrosine kinase, typically results in phosphorylation of the receptor. The adaptor protein Grb2 associates with the phosphorylated intracellular domain of the activated receptor, and this association recruits guanine nucleotide exchange factors including SOS-I and CDC25 to the cell membrane. These guanine nucleotide exchange factors interact with and activate the GTPase Ras. Common Ras isoforms include K-Ras, N-Ras, H-Ras and others. Following Ras activation, the serine/threonine kinase Raf (e.g., A-Raf, B-Raf or Raf-1 ) is recruited to the cell membrane through interaction with Ras. Raf is then phosphorylated. Raf directly activates MEKI and MEK2 by phosphorylation of two serine residues at positions 217 and 221 . Following activation, MEKI and MEK2 phosphorylate tyrosine (Tyr-185) and threonine (Thr-183) residues in serine/threonine kinases Erkl and Erk2, resulting in Erk activation. Activated Erk regulates many targets in the cytosol and also translocates to the nucleus, where it phosphorylates a number of transcription factors regulating gene expression. Erk kinase has numerous targets, including Elk-I, c-Etsl, c-Ets2, p90RSKI, MNKI, MNK2, MSKI, MSK2 and TOB. While the foregoing pathway is a classical representation of MAPK signaling, there is considerable cross talk between the MAPK pathway and other signaling cascades.
[0040] Aberrations in MAPK signaling have a significant role in cancer biology.
Altered expression of Ras is common in many cancers, and activating mutations in Ras have also been identified. Such mutations are found in up to 30% of all cancers, and are especially common in pancreatic (90%) and colon (50%) carcinomas. In addition, activating Raf mutations have been identified in melanoma and ovarian cancer. The most common mutation, BRAFV600E, results in constitutive activation of the downstream MAP kinase pathway and is required for melanoma cell proliferation, soft agar growth, and tumor xenograft formation. Based on the defined role of MAPK over-activation in human cancers, targeting components of the MAPK pathway with specific inhibitors is a promising approach to cancer therapy. However, patients may have innate resistance or acquire resistance to these promising therapies. Identification of target kinases, diagnostic and/or prognostic markers and treatment therapies for these patients with innate or acquired resistance are described below.
[0041] High Throughput Functional Screening Assay
[0042] In some aspects, the present invention relates to methods of identifying targets capable of driving resistance to clinically efficacious therapies using a high throughput screening assay. In some embodiments, the method may include an open reading frame (ORF)-based functional screen for kinases that drive resistance to therapeutic agents. The method may include providing a cell line with a kinase known to have an oncogenic mutation. A library of kinase ORFs may be individually expressed in the cell line so that a plurality of clones each expressing a different ORF from the library may be further evaluated. Each clone may be (1 ) exposed to an inhibitor of the known kinase in the cell line and (2) monitored for growth changes based on the expression of the ORF in the cell line without the inhibitor. Any clones having a growth effect from the ORF expression alone, either positive or negative growth, are eliminated. The remaining clones each expressing a different kinase are then compared for viability between a control and a treated clone and normalized to a positive control. Increased cell viability after treatment with the inhibitor identifies ORFs that confer resistance and therefore identifies kinase targets for treatment with an additional inhibitor. In some embodiments, clones scoring above two standard deviations from the normalized mean may be target kinases indicating treatment with an additional inhibitor is beneficial to the subject.
[0043] By way of non-limiting example, a schematic of a high throughput functional screening assay for kinases that drive resistance to B-RAF inhibition is shown in Figure 5. A collection of -600 cloned and sequence validated ORFs were assembled, accounting for -75% of all annotated kinases (Center for Cancer Systems Biology (CCSB)/Broad Institute Kinase ORF Collection, Figs. 1 a, 1 b, Table 3). This publically available collection can be rapidly transferred into a variety of expression vectors for various end-applications. Any type of expression vector known to one skilled in the art may be used to express the Kinase ORF collection. By way of non-limiting example, a selectable, epitope-tagged, lentiviral expression vector capable of producing high titer virus and robust ORF expression in mammalian cells may be created to express the kinase collection, (pLX-BLAST-V5, Figure 6a).
[0044] To identify kinases capable of circumventing RAF inhibition, the arrayed kinase ORF collection may be stably expressed in A375, a B-RAFV600E malignant melanoma cell line that is sensitive to the RAF kinase inhibitor PLX4720 (Figs 1 a, 1 b and 6c, Table 3). Clones of ORF expressing cells treated with 1 μΜ PLX4720 are screened for viability relative to untreated cells and normalized to an assay-specific positive control, MEK1 S218 2220 (MEK1 DD) (Table 4). ORFs that affected baseline viability or proliferation are removed from the analysis. Clones scoring above two standard deviations from the normalized mean may be further evaluated to identify a resistance conferring target kinase for a second inhibitor. In some embodiments, the gene encoding the target kinase may be MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL(CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3). In some embodiments, the gene encoding the target kinase may be a MAPK pathway activator. In some embodiments, the gene encoding the target kinase may be a MAP3 kinase that directly phosphorylates and activates MEK. In some embodiments, the gene encoding the target kinase may encode an adapter protein that is amplified and phosphorylated in melanoma. [0045] In other embodiments, the ORF collection may be stably expressed in a cell line having a different mutation in B-RAF, for example, another mutation at about amino acid position 600 such as V600K, V600D, and V600R. Additional B-RAF mutations include the mutations described in Davies et al. Nature, 417, 949-954, 2002, Table 1 . Cell lines may be used that are sensitive to other RAF kinase inhibitors including, but not limited to, PLX4032; GDC-0879; RAF265; sorafenib; SB590855 and/or ZM 336372. In some embodiments, the ORF collection may be stably expressed in a cell line having a sensitivity to a MEK inhibitor. Non-limiting examples of MEK inhibitors include, AZD6244; CI-1040; PD184352; PD318088, PD98059, PD334581 , RDEA1 19, 6- Methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinoline-3- carbonitrile and 4-[3-Chloro-4-(1 -methyl-1 H-imidazol-2-ylsulfanyl)-phenylamino]-6- methoxy-7-(3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrile. Additional RAF and MEK inhibitors are described below. By way of non-limiting example, exemplary RAF inhibitors are shown in Table 1 and exemplary MEK inhibitors are shown in Table 2.
[0046] Table 1 : Exemplary RAF Inhibitors
47] Table 2: Exemplary MEK Inhibitors
Name CAS No. Structure
[0048] Diagnostic/Prognostic Markers for Innate and Acquired Resistance to Targeted Therapies
[0049] In some aspects, the present invention relates to methods of detecting the presence of one or more diagnostic or prognostic markers in a sample (e.g. a biological sample from a cancer patient). A variety of screening methods known to one of skill in the art may be used to detect the presence of the marker in the sample including DNA, RNA and protein detection. The techniques described below can be used to determine the presence or absence of a kinase target in a sample obtained from a patient. In some embodiments, the patient may have innate or acquired resistance to kinase targeted therapies, including B-RAF inhibitors or MEK inhibitors. For example, the patient may have an innate or acquired resistance to B-RAF inhibitors PLX4720 and/or PLX4032. In some embodiments, the patient may have innate or acquired resistance to MEK inhibitor AZD6244. Identification of one or more kinase targets markers in a patient assists the physician in determining a treatment protocol for the patient. For example, in a patient having one or more kinase target markers, the physician may treat the patient with a combination therapy as described in more detail below.
[0050] In some embodiments, the kinase target may include, but is not limited to, MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL(CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3). The marker may be an increase in the gene copy number, an increase in protein expression, phosphorylation of one or more MAP kinase pathway members, a change in mRNA expression and the like, for the kinase target.
[0051] By way of non-limiting example, in a patient having an oncogenic mutation in
B-RAF, identification of an activated target kinase can be useful for characterizing a treatment protocol for the patient. For example, in a patient having a B-RAFV600E mutation, treatment with a RAF inhibitor alone may indicate that the patient is at relatively high risk of acquiring resistance to the treatment after a period of time. In a patient having an oncogenic mutation, identification of an activated kinase target in that patient may indicate inclusion of a second inhibitor in the treatment protocol.
[0052] Identification of an activated kinase target may include an analysis of a gene copy number and identification of an increase in copy number of a target kinase. For example, a copy number gain in MAP3K8 is indicative of a patient having innate resistance or developing acquired resistance, in particular if the patient also has a B- RAFV6OOE M UTATION
[0053] In some embodiments, identification of an activated kinase target may include an analysis of phosphorylation of a kinase target and/or a member of the MAP kinase pathway. For example, phosphorylation of C-RAF at S338 is indicative of a patient having innate resistance or developing acquired resistance, in particular if the patient also has a B-RAFV600E mutation. In some embodiments, identification of an increase in MEK/ERK phosphorylation may be indicative of a patient having innate resistance or developing acquired resistance. Increased COT protein expression in patients having a B-RAFV600E mutation may predict resistance to RAF inhibition and MEK inhibition.
[0054] Identification of an activated kinase target may include an analysis of mRNA expression of a kinase target. For example, an increase in COT mRNA expression following initial treatment with a first kinase inhibitor is indicative of a patient having or developing resistance. In some embodiments, the first kinase inhibitor may be a RAF inhibitor or a MEK inhibitor.
[0055] Methods of treatment
[0056] In various embodiments, the invention provides methods for treatment of a patient having cancer. The methods generally comprise administration of a first inhibitor and a second inhibitor. One inhibitor may be a RAF inhibitor. The RAF inhibitor may be a pan-RAF inhibitor or a selective RAF inhibitor. Pan-RAF inhibitors include but are not limited to RAF265, sorafenib, or SB590885. In some embodiments, the RAF inhibitor is a B-RAF inhibitor. In some embodiments, the selective RAF inhibitor is PLX4720, PLX4032, or GDC-0879-A. One inhibitor may be a MEK inhibitor (see Table 2 illustrating exemplary MEK inhibitors). One inhibitor may be a COT inhibitor. By way of non-limiting example, the COT inhibitor may be a shRNA inhibitor as described below or a small molecule COT inhibitor, 4-(3-chloro-4-fluorophenylamino)-6-(pyridin-3-yl- methylamino)-3-cyano-[1 ,7]-naphthyridine (EMD;TPL2 inhibitor I; catalogue number 616373, PubChem ID: 9549300) Inhibitors of the present invention inhibit one or more of the kinase targets including MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL(CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3) or other MAP kinase pathway targets.
[0057] In some embodiments, a combination therapy for cancer is provided, comprising an effective amount of a RAF inhibitor and an effective amount of a MAP3K8 (TPL2/COT) inhibitor. Also provided herein is a combination therapy for cancer, comprising an effective amount of a RAF inhibitor and an effective amount of a MEK inhibitor. Other combination therapies include an effective amount of a RAF inhibitor and an effective amount of a second inhibitor targeting the gene, mRNA or protein encoded by one or more of the following: MAP3K8 (TPL2/COT), RAF1 (CRAF),
CRKL(CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch) , ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3). The combination therapy is suitable for treatment of a patient wherein the cancer contains B-RAF mutant cells and in particular, B-RAFV600E mutant cells. The present invention further provides a combination therapy for cancer, comprising an effective amount of a RAF inhibitor and an effective amount of a MEK inhibitor, wherein the subject with the cancer contains cells with altered MAP3K8 (TPL2/COT) expression or gene copy number. In some embodiments, the MEK inhibitor is CI-1040/PD184352 or AZD6244.
[0058] As a non-limiting example, the MEK inhibitor provided herein can be Cl- 1040, AZD6244, PD318088, PD98059, PD334581 , RDEA1 19, 6-Methoxy-7-(3- morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)-quinoline-3-carbonitrile or 4-[3- Chloro-4-(1 -methyl-1 H-imidazol-2-ylsulfanyl)-phenylamino]-6-methoxy-7-(3-morpholin-4- yl-propoxy)-quinoline-3-carbonitrile, Roche compound RG7420, or combinations thereof. Additional MEK inhibitors known in the art may also be used.
[0059] In exemplary embodiments of the foregoing aspects, the RAF inhibitor provided herein is PLX4720, PLX4032, BAY 43-9006 (Sorafenib), ZM 336372, RAF 265, AAL-881 , LBT-613, or CJS352 (NVP-AAL881 -NX (hereafter referred to as
AAL881 ) and NVP-LBT613-AG-8 (LBT613) are isoquinoline compounds
(Novartis, Cambridge, MA). Additional exemplary RAF inhibitors useful for
combination therapy include pan-RAF inhibitors, inhibitors of B-RAF, inhibitors of A- RAF, and inhibitors of RAF-1 . In exemplary embodiments RAF inhibitors useful for combination therapy include PLX4720, PLX4032, BAY 43-9006 (Sorafenib), ZM 336372, RAF 265, AAL-881 , LBT-613, and CJS352. Exemplary RAF inhibitors further include the compounds set forth in PCT Publication No. WO/2008/028141 , the entire contents of which are incorporated herein by reference. Exemplary RAF inhibitors additionally include the quinazolinone derivatives described in PCT
Publication No. WO/2006/024836, and the pyridinylquinazolinamine derivatives described in PCT Publication No. WO/2008/020203, the entire contents of which are incorporated herein by reference.
[0060] Administration of the combination includes administration of the combination in a single formulation or unit dosage form, administration of the individual agents of the combination concurrently but separately, or administration of the individual agents of the combination sequentially by any suitable route. The dosage of the individual agents of the combination may require more frequent administration of one of the agents as compared to the other agent in the combination. Therefore, to permit appropriate dosing, packaged pharmaceutical products may contain one or more dosage forms that contain the combination of agents, and one or more dosage forms that contain one of the combinations of agents, but not the other agent(s) of the combination.
[0061] Agents may contain one or more asymmetric elements such as stereogenic centers or stereogenic axes, e.g., asymmetric carbon atoms, so that the compounds can exist in different stereoisomeric forms. These compounds can be, for example, racemates or optically active forms. For compounds with two or more asymmetric elements, these compounds can additionally be mixtures of diastereomers. For compounds having asymmetric centers, it should be understood that all of the optical isomers and mixtures thereof are encompassed. In addition, compounds with carbon- carbon double bonds may occur in Z- and E-forms; all isomeric forms of the compounds are included in the present invention. In these situations, the single enantiomers (optically active forms) can be obtained by asymmetric synthesis, synthesis from optically pure precursors, or by resolution of the racemates. Resolution of the
racemates can also be accomplished, for example, by conventional methods such as crystallization in the presence of a resolving agent, or chromatography, using, for example a chiral HPLC column.
[0062] Unless otherwise specified, or clearly indicated by the text, reference to compounds useful in the combination therapy of the invention includes both the free base of the compounds, and all pharmaceutically acceptable salts of the compounds. A preferred salt is the hydrochloride salt.
[0063] The term "pharmaceutically acceptable salts" includes derivatives of the disclosed compounds, wherein the parent compound is modified by making non-toxic acid or base addition salts thereof, and further refers to pharmaceutically acceptable solvates, including hydrates, of such compounds and such salts. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid addition salts of basic residues such as amines; alkali or organic addition salts of acidic residues such as carboxylic acids; and the like, and combinations comprising one or more of the foregoing salts. The pharmaceutically acceptable salts include non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, and nitric; other acceptable inorganic salts include metal salts such as sodium salt, potassium salt, and cesium salt; and alkaline earth metal salts, such as calcium salt and magnesium salt; and combinations comprising one or more of the foregoing salts.
[0064] Pharmaceutically acceptable organic salts include salts prepared from organic acids such as acetic, trifluoroacetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,
HOOC(CH2)nCOOH where n is 0-4; organic amine salts such as triethylamine salt, pyridine salt, picoline salt, ethanolamine salt, triethanolamine salt, dicyclohexylamine salt, Ν,Ν'-dibenzylethylenediamine salt; and amino acid salts such as arginate, asparginate, and glutamate, and combinations comprising one or more of the foregoing salts.
[0065] An "effective amount" of a combination of agents (e.g., MEK and RAF inhibitors, or RAF and COT inhibitors, or RAF and an inhibitor targeting MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL(CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3)) is an amount sufficient to provide an observable improvement over the baseline clinically observable signs and symptoms of the disorder treated with the combination.
[0066] The pharmaceutical products can be administrated by oral or other forms, e.g., rectally or by parenteral injection. "Oral dosage form" is meant to include a unit dosage form prescribed or intended for oral administration. An oral dosage form may or may not comprise a plurality of subunits such as, for example, microcapsules or microtablets, packaged for administration in a single dose.
[0067] The pharmaceutical products can be released in various forms. "Releasable form" is meant to include instant release, immediate-release, controlled-release, and sustained-release forms.
[0068] "Instant-release" is meant to include a dosage form designed to ensure rapid dissolution of the active agent by modifying the normal crystal form of the active agent to obtain a more rapid dissolution.
[0069] "Immediate-release" is meant to include a conventional or non-modified release form in which greater than or equal to about 50% or more preferably about 75% of the active agents is released within two hours of administration, preferably within one hour of administration.
[0070] "Sustained-release" or "extended-release" includes the release of active agents at such a rate that blood (e.g., plasma) levels are maintained within a therapeutic range but below toxic levels for at least about 8 hours, preferably at least about 12 hours, more preferably about 24 hours after administration at steady-state. The term "steady-state" means that a plasma level for a given active agent or
combination of active agents, has been achieved and which is maintained with subsequent doses of the active agent(s) at a level which is at or above the minimum effective therapeutic level and is below the minimum toxic plasma level for a given active agent(s).
[0071] The term "treat", "treated," "treating" or "treatment" is used herein to mean to relieve, reduce or alleviate at least one symptom of a disease in a subject. For example, treatment can be diminishment of one or several symptoms of a disorder or complete eradication of a disorder, such as cancer. Within the meaning of the present invention, the term "treat" also denote to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease. The term "protect" is used herein to mean prevent delay or treat, or all, as appropriate, development or continuance or aggravation of a disease in a subject.
Within the meaning of the present invention, the disease is associated with a cancer.
[0072] The term "subject" or "patient" is intended to include animals, which are capable of suffering from or afflicted with a cancer or any disorder involving, directly or indirectly, a cancer. Examples of subjects include mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In certain embodiments, the subject is a human, e.g., a human suffering from, at risk of suffering from, or potentially capable of suffering from cancers.
[0073] The term "about" or "approximately" usually means within 20%, more preferably within 10%, and most preferably still within 5% of a given value or range. Alternatively, especially in biological systems, the term "about" means within about a log (i.e., an order of magnitude) preferably within a factor of two of a given value.
[0074] The use of the terms "a" and "an" and "the" and similar referents in the context of describing the invention (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising, "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.
[0075] As specified above, in one aspect, the instant invention provides a drug combination useful for treating, preventing, arresting, delaying the onset of and/or reducing the risk of developing, or reversing at least one symptom of cancer, in a subject comprising administering to the subject a combination therapy, comprising an effective amount of a RAF inhibitor and an effective amount of a MAP3K8 (TPL2/COT) inhibitor, or an effective amount of a RAF inhibitor and an effective amount of MEK inhibitor or an effective amount of a RAF inhibitor and an effective amount of a second inhibitor targeting MAP3K8 (TPL2/COT), RAF1 (CRAF), CRKL(CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3).
Preferably, these inhibitors are administered at therapeutically effective dosages which, when combined, provide a beneficial effect. The administration may be simultaneous or sequential.
[0076] The term "cancer" is used herein to mean a broad spectrum of tumors, including all solid tumors and hematological malignancies. Examples of such tumors include but are not limited to leukemias, lymphomas, myelomas, carcinomas, metastatic carcinomas, sarcomas, adenomas, nervous system cancers and geritourinary cancers. In exemplary embodiments, the foregoing methods are useful in treating adult and pediatric acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, anal cancer, cancer of the appendix, astrocytoma, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancer, osteosarcoma, fibrous histiocytoma, brain cancer, brain stem glioma, cerebellar astrocytoma, malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, hypothalamic glioma, breast cancer, male breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoid tumor, carcinoma of unknown origin, central nervous system lymphoma, cerebellar astrocytoma, malignant glioma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, Ewing family tumors, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal stromal tumor, extracranial germ cell tumor, extragonadal germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor, glioma, hairy cell leukemia, head and neck cancer, hepatocellular cancer, Hodgkin lymphoma, non-Hodgkin lymphoma,
hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular
melanoma, islet cell tumors, Kaposi sarcoma, kidney cancer, renal cell cancer, laryngeal cancer, lip and oral cavity cancer, small cell lung cancer, non-small cell lung cancer, primary central nervous system lymphoma, Waldenstrom macroglobulinema, malignant fibrous histiocytoma, medulloblastoma, melanoma, Merkel cell carcinoma, malignant mesothelioma, squamous neck cancer, multiple endocrine neoplasia syndrome, multiple myeloma, mycosis fungoides, myelodysplastic syndromes, myeloproliferative disorders, chronic myeloproliferative disorders, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive
neuroectodermal tumors, pituitary cancer, plasma cell neoplasms, pleuropulmonary blastoma, prostate cancer, rectal cancer, rhabdomyosarcoma, salivary gland cancer, soft tissue sarcoma, uterine sarcoma, Sezary syndrome, non-melanoma skin cancer, small intestine cancer, squamous cell carcinoma, squamous neck cancer, supratentorial primitive neuroectodermal tumors, testicular cancer, throat cancer, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer, trophoblastic tumors, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, and Wilms tumor.
[0077] In particular, the cancer may be associated with a mutation in the B-RAF gene. These cancers include melanoma, breast cancer, colorectal cancers, glioma, lung cancer, ovarian cancer, sarcoma and thyroid cancer.
[0078] In a particular embodiment, the therapeutic combination provided herein is effective for the treatment of moderate to severe cancer in a subject.
[0079] Dosages [0080] The optimal dose of the combination of agents for treatment of cancer can be determined empirically for each subject using known methods and will depend upon a variety of factors, including the activity of the agents; the age, body weight, general health, gender and diet of the subject; the time and route of administration; and other medications the subject is taking. Optimal dosages may be established using routine testing and procedures that are well known in the art.
[0081] The amount of combination of agents that may be combined with the carrier materials to produce a single dosage form will vary depending upon the individual treated and the particular mode of administration. In some embodiments the unit dosage forms containing the combination of agents as described herein will contain the amounts of each agent of the combination that are typically administered when the agents are administered alone.
[0082] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the
compounds of the invention employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
[0083] In general, a suitable daily dose of a compound of the invention will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. Such an effective dose will generally depend upon the factors described above and is readily determined by one having skill in the art.
[0084] Generally, therapeutically effective doses of the compounds of this invention for a patient, when used for the indicated analgesic effects, will range from about 0.0001 to about 1000 mg per kilogram of body weight per day, more preferably from about 0.01 to about 50 mg per kg per day.
[0085] If desired, the effective daily dose of the active compound may be
administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
[0086] Pharmaceutical Formulations and Routes of Administration [0087] Provided herein are pharmaceutical formulations comprising a combination of agents for the treatment of cancer, e.g., melanoma. The pharmaceutical formulations may additionally comprise a carrier or excipient, stabilizer, flavoring agent, and/or coloring agent.
[0088] Provided herein are pharmaceutical formulations comprising combination of agents which can be, for example, a combination of two types of agents: (1 ) a RAF inhibitor and/or pharmacologically active metabolites, salts, solvates and racemates of the inhibitor and (2) a MAP3K8 (TPL2/COT) inhibitor and/or pharmacologically active metabolites, salts, solvates and racemates of the COT inhibitor. In another embodiment the combination of agents may be provided for a subject comprising BRAF mutant cells or comprising cells over expressing MAP3K8 (TPL2/COT) and include: (1 ) a RAF inhibitor and/or pharmacologically active metabolites, salts, solvates and racemates of the inhibitor and (2) a MEK inhibitor and/or pharmacologically active metabolites, salts, solvates and racemates of the MEK inhibitor.
[0089] The combination of agents may be administered using a variety of routes of administration known to those skilled in the art. The combination of agents may be administered to humans and other animals orally, parenterally, sublingually, by aerosol ization or inhalation spray, rectally, intracisternally, intravaginally,
intraperitoneally, bucally, or topically in dosage unit formulations containing
conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may also involve the use of transdermal administration such as transdermal patches or ionophoresis devices. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.
[0090] Methods of formulation are well known in the art and are disclosed, for example, in Remington: The Science and Practice of Pharmacy, Mack Publishing Company, Easton, Pa., 19th Edition (1995). Pharmaceutical compositions for use in the present invention can be in the form of sterile, non-pyrogenic liquid solutions or suspensions, coated capsules, suppositories, lyophilized powders, transdermal patches or other forms known in the art. [0091] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution, suspension or emulsion in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1 ,3 propanediol or 1 ,3 butanediol.
Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S. P. and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono or di glycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. The injectable formulations can be sterilized, for example, by filtration through a bacterial-retaining filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
[0092] In order to prolong the effect of a drug, it is often desirable to slow the absorption of the drug from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form.
Alternatively, delayed absorption of a parenterally administered drug form may be accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations may also be prepared by entrapping the drug in liposomes or microemulsions, which are compatible with body tissues.
[0093] Compositions for rectal or vaginal administration are preferably suppositories which can be prepared by mixing the compounds of this invention with suitable non irritating excipients or carriers such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active compound.
[0094] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert, pharmaceutically acceptable excipient or carrier such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders such as, for example,
carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium
carbonate, e) solution retarding agents such as paraffin, f) absorption accelerators such as quaternary ammonium compounds, g) wetting agents such as, for example, acetyl alcohol and glycerol monostearate, h) absorbents such as kaolin and bentonite clay, and i) lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may also comprise buffering agents.
[0095] Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
[0096] The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.
[0097] The active compounds can also be in micro-encapsulated form with one or more excipients as noted above. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings, release controlling coatings and other coatings well known in the pharmaceutical formulating art. In such solid dosage forms the active compound may be admixed with at least one inert diluent such as sucrose, lactose or starch. Such dosage forms may also comprise, as is normal practice, additional substances other than inert diluents, e.g., tableting lubricants and other tableting aids such a magnesium stearate and microcrystalline cellulose. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. They may optionally contain opacifying agents and can also be of a composition that they release the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.
[0098] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, EtOAc, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3 butylene glycol,
dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, the oral compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.
[0099] Dosage forms for topical or transdermal administration of a compound of this invention include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants or patches. The active component is admixed under sterile conditions with a pharmaceutically acceptable carrier and any needed preservatives or buffers as may be required. Ophthalmic formulations, ear drops, and the like are also contemplated as being within the scope of this invention.
[00100] The ointments, pastes, creams and gels may contain, in addition to an active compound of this invention, excipients such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicic acid, talc and zinc oxide, or mixtures thereof. [00101] Compositions of the invention may also be formulated for delivery as a liquid aerosol or inhalable dry powder. Liquid aerosol formulations may be nebulized predominantly into particle sizes that can be delivered to the terminal and respiratory bronchioles.
[00102] Aerosolized formulations of the invention may be delivered using an aerosol forming device, such as a jet, vibrating porous plate or ultrasonic nebulizer, preferably selected to allow the formation of an aerosol particles having with a mass medium average diameter predominantly between 1 to 5 m. Further, the formulation preferably has balanced osmolarity ionic strength and chloride concentration, and the smallest aerosolizable volume able to deliver effective dose of the compounds of the invention to the site of the infection. Additionally, the aerosolized formulation preferably does not impair negatively the functionality of the airways and does not cause undesirable side effects.
[00103] Aerosol ization devices suitable for administration of aerosol formulations of the invention include, for example, jet, vibrating porous plate, ultrasonic nebulizers and energized dry powder inhalers, that are able to nebulize the formulation of the invention into aerosol particle size predominantly in the size range from 1 5 m.
Predominantly in this application means that at least 70% but preferably more than 90% of all generated aerosol particles are within 1 5 m range. A jet nebulizer works by air pressure to break a liquid solution into aerosol droplets. Vibrating porous plate nebulizers work by using a sonic vacuum produced by a rapidly vibrating porous plate to extrude a solvent droplet through a porous plate. An ultrasonic nebulizer works by a piezoelectric crystal that shears a liquid into small aerosol droplets. A variety of suitable devices are available, including, for example, AERONEB and AERODOSE vibrating porous plate nebulizers (AeroGen, Inc., Sunnyvale, California), SIDESTREAM nebulizers (Medic Aid Ltd., West Sussex, England), PARI LC and PARI LC STAR jet nebulizers (Pari Respiratory Equipment, Inc., Richmond, Virginia), and AEROSONIC (DeVilbiss Medizinische Produkte (Deutschland) GmbH, Heiden, Germany) and
ULTRAAIRE (Omron Healthcare, Inc., Vernon Hills, Illinois) ultrasonic nebulizers.
[00104] Compounds of the invention may also be formulated for use as topical powders and sprays that can contain, in addition to the compounds of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays can additionally contain customary propellants such as chlorofluorohydrocarbons.
[00105] Transdermal patches have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms can be made by dissolving or dispensing the compound in the proper medium. Absorption enhancers can also be used to increase the flux of the compound across the skin. The rate can be controlled by either providing a rate controlling membrane or by dispersing the compound in a polymer matrix or gel. The compounds of the present invention can also be
administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono or multi lamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain, in addition to a compound of the present invention, stabilizers, preservatives, excipients, and the like. The preferred lipids are the phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic. Methods to form liposomes are known in the art. See, for example, Prescott (ed.), "Methods in Cell Biology," Volume XIV, Academic Press, New York, 1976, p. 33 et seq.
[00106] Examples
[00107] Example 1 : An ORF-based functional screen identifies specific kinases as drivers of resistance to B-RAF inhibition.
[00108] To identify kinases capable of circumventing RAF inhibition, 597 sequence-validated kinase ORF clones representing -75% of annotated kinases (Center for Cancer Systems Biology (CCSB)/Broad Institute Kinase ORF Collection) were assembled and stably expressed in A375, a B-RAFV600E malignant melanoma cell line that is sensitive to the RAF kinase inhibitor PLX4720 (Tsai, J. et al. Proc. Natl Acad. Sci. USA 105, 3041-3046 (2008)) (Fig. 1 a, 1 b, Table 3, Fig. 6c). ORF expressing cells treated with 1 μΜ PLX4720 were screened for viability relative to untreated cells and normalized to an assay-specific positive control, MEK1S218 222D (MEK1 DD) (Emery, C. M. et al. Proc. Natl Acad. Sci. USA 106, 2041 1-20416 (2009).) (Table 4 and summarized in Fig. 5). Nine ORFs conferred resistance at levels exceeding two standard deviations from the mean (Fig. 1 b and Table 4) and were selected for follow-up analysis (Fig. 7). Three of nine candidate ORFs were receptor tyrosine kinases, underscoring the potential of this class of kinases to engage resistance pathways. Resistance effects were validated and prioritized across a multi-point PLX4720 drug concentration scale in the B-RAFV600E cell lines A375 and SKMEL28. The Ser/Thr MAP kinase kinase kinases (MAP3Ks) MAP3K8 (COT/Tpl2) and RAF1 (C-RAF) emerged as top candidates from both cell lines; these ORFs shifted the PLX4720 Gl50 by 10-600 fold without affecting viability (Table 5 and Figs. 8 and 9). CRKL, an ORF that shifted the PLX4720 Gl50 to a lesser extent (9.7 fold in SKMEL28 cells; Fig. 8), encodes an adapter protein phosphorylated by tyrosine kinases such as BCR-ABL (Birge, R.B. et al., Cell Commun Signal 7, 13 (2009)), but lacks intrinsic kinase activity. COT and C-RAF reduced sensitivity to PLX4720 in multiple B-RAFV600E cell lines (Fig. 1c) confirming the ability of these kinases to mediate resistance to RAF inhibition. A secondary screen in A375 and SKMEL28 prioritizes the top 9 candidate ORFs across a multipoint PLX4720 concentration scale (Fig. 1 d).
[00109] Interestingly, the top two validated kinases are both Ser/Thr MAP kinase kinase kinases (MAP3Ks) known to activate MEK/ERK signaling in several contexts. Like B-RAF, C-RAF is a MAP3K in the canonical MAPK cascade (McKay, M.M. and Morrison, D.K. Oncogene 26, 31 13-3121 (2007)) that was previously implicated in resistance associated with stepwise selection in vitro using a pan-RAF inhibitor
(Montagut, C. et al. Cancer Res 68, 4853-4861 (2008)). COT (the protein product of the human MAP3K8 gene) is best characterized as the MAP3K (Salmeron, A. et al. EMBO J 15, 817-826 (1996)) downstream of NFKB signaling in inflammatory cells (Banerjee, A. et al., Proc Natl Acad Sci U.S.A. 103, 3274-3279 (2006)); however, its functional importance in human cancer has not previously been elucidated.
[00110] Example 2: Resistance to B-RAF inhibition via MAPK pathway activation.
[00111] Whether the overexpression of these genes was sufficient to activate the MAPK pathway was also tested. At baseline, COT expression increased ERK phosphorylation in a manner comparable to MEK1 , consistent with MAP kinase pathway activation (Figs. 2a and 10). Overexpression of wild-type COT or C-RAF resulted in constitutive phosphorylation of ERK and MEK in the presence of
PLX4720, whereas kinase-dead derivatives had no effect (Figs. 2a and 1 1 ). Thus, COT and C-RAF drive resistance to RAF inhibition predominantly through
re-activation of MAPK signaling. Notably, of the nine candidate ORFs from the initial screen, a subset (3) did not show persistent ERK/MEK phosphorylation following RAF inhibition, suggesting MAPK pathway-independent alteration of drug sensitivity (Figure 12).
[00112] Example 3: C-RAF activation and heterodimerization with B-RAF.
[00113] C-RAF activation and heterodimerization with B-RAF constitute critical components of the cellular response to B-RAF inhibition. In A375 cells, endogenous C-RAF: B-RAF heterodimers were measurable and inducible following treatment with PLX4720 (Fig. 13). However, endogenous C-RAF phosphorylation at S338— an event required for C-RAF activation— remained low (Fig. 13). In contrast, ectopically
expressed C-RAF was phosphorylated on S338 (Fig. 13) and its PLX4720 resistance phenotype was associated with sustained MEK/ERK activation (Figs. 2a and 13).
Moreover, ectopic expression of a high-activity C-RAF truncation mutant (C-RAF(W22) was more effective than wild-type C-RAF in mediating PLX4720 resistance and ERK activation (Fig. 14), further indicating that elevated C-RAF activity directs resistance to this agent. Consistent with this model, oncogenic alleles of NRAS and KRAS conferred PLX4720 resistance in A375 cells (Fig. 2b) and yielded sustained C-RAF(S338) and ERK phosphorylation in the context of drug treatment (Fig. 2c). Thus, although genetic alterations that engender C-RAF activation (e.g., oncogenic RAS mutations) tend to show mutual exclusivity with B-RAFV600E mutation, such co-occurring events are favored in the context of acquired resistance to B-RAF inhibition.
[00114] Example 4: Investigation of COT expression in Melanoma
[00115] While C-RAF has been linked previously to melanoma and MAPK pathway dependencies (Montagut, C. et al. 2008; Karreth, F.A., DeNicola, G.M. et al., 2009; Dumaz, N. et al. Cancer Res 66, 9483-9491 (2006); Hatzivassiliou, G. et al. Nature (2010); Heidorn, S.J. et al., Cell 140, 209-221 (2010); Poulikakos, P.I. et al., Nature (2010)), COT has not been described as a melanoma-associated kinase.
[00116] The role of COT in melanoma was investigated, and its expression in human melanocytes was examined. Primary immortalized melanocytes (B-RAF wild-type) expressed COT (Fig. 2d), although ectopic B-RAFV600E expression reduced COT mRNA levels (Fig. 15) and rendered COT protein undetectable (Fig. 2d).
Conversely, whereas ectopically expressed COT was only weakly detectable in A375 cells (Figs. 2a, 2e), shRNA-mediated depletion of endogenous B-RAFV600E caused an increase in COT protein levels that correlated with the extent of B-RAF knockdown (Fig. 2e). Moreover, treatment of COT-expressing A375 cells with PLX4720 led to a dose-dependent increase in COT protein (Fig. 2a) without affecting ectopic COT mRNA levels (Fig. 15). Oncogenic B-RAF antagonizes COT expression largely through altered protein stability (Figs. 2a, d, e, and 15), and B-RAF inhibition potentiates the outgrowth of COT-expressing cells during the course of treatment. Notably, neither C-RAF nor B-RAF alone or in combination was required for ERK phosphorylation in the context of COT expression, even in the presence of PLX4720 (Figs. 2e, 2f and Fig. 16). As shown, COT expression is sufficient to induce MAP kinase pathway activation in a RAF-independent manner.
[00117] Example 5: COT expression predicts resistance to B-RAF inhibition in cancer cell lines.
[00118] Whether cell lines expressing elevated COT in a B-RAFV600E background exhibit de novo resistance to PLX4720 treatment was tested. To identify such instances, a panel of cell lines was screened for evidence of MAP3K8/COT copy number gains coincident with the B-RAFV600E mutation. Of 534 cell lines that had undergone copy number analysis and mutation profiling, 38 cell lines (7.1 %) contained the B-RAFV600E mutation. Within this subgroup, two cell lines— OUMS-23 (colon cancer) and RPMI-7951 (melanoma)— also showed evidence of chromosomal copy gains spanning the MAP3K8/COT locus (Figs. 3a and 17) and robust COT protein expression (Figs. 3b and 18). A panel of melanoma short-term cultures was also screened for COT protein expression. One of these lines expressed COT: M307, a short-term culture derived from a B-RAFV600E tumor that developed resistance to allosteric MEK inhibition following initial disease stabilization (Fig. 3c). All three cell lines were refractory to PLX4720 treatment, exhibiting GI50 values in the range of 8-10 μΜ (Fig. 3d) and showing sustained ERK phosphorylation in the context of B-RAF inhibition (Figs. 3e, 3f). OUMS-23 and RPMI-7951 are MAPK pathway inhibitor-na'fve cell lines; thus, these results demonstrate that COT confers de novo resistance to RAF inhibition (a
phenomenon observed in -10% of B-RAFV600E melanomas).
[00119] Example 6: COT expression in patients treated with a RAF inhibitor.
[00120] COT expression in the context of resistance to the clinical RAF inhibitor PLX4032 was examined by obtaining biopsy material from 3 patients with metastatic, B-RAFV600E melanoma. Each case consisted of frozen, lesion-matched biopsy material obtained prior to and during treatment ("pre-treatment" and "on-treatment"; Fig. 3g, Table 6); additionally, one sample contained two independent biopsy specimens from the same relapsing tumor site ("post-relapse"; Fig. 3g). Consistent with the
experimental models presented above, quantitative real-time RT-PCR (qRT/PCR) analysis revealed increased COT mRNA expression concurrent with PLX4032 treatment in 2 of 3 cases. COT mRNA levels were further increased in a relapsing specimen relative to its pre-treatment and on-treatment counterparts (Fig. 3g, Patient #1 ). An additional, unmatched relapsed malignant melanoma biopsy showed elevated COT mRNA expression comparable to levels observed in RAF inhibitor-resistant, COT-amplified cell lines (Fig. 19). This specimen also exhibited robust MAPK pathway activation and elevated expression of B-RAF, C-RAF and COT relative to matched normal skin or B-RAFV600E cell lines (Fig. 19). Sequencing studies of this tumor revealed no additional mutations in BRAF, NRAS or KRAS (data not shown). These analyses provided clinical evidence that COT-dependent mechanisms are operant in PLX4032-resistant malignant melanomas.
[00121] Example 7: COT regulation of MEK/ERK phosphorylation.
[00122] Whether COT actively regulates MEK/ERK phosphorylation in B-RAFV600E cells that harbor naturally elevated COT expression was tested by introducing shRNA constructs targeting COT into RPMI-7951 cells. Depletion of COT suppressed
RPMI-7951 viability (Fig. 20) and decreased ERK phosphorylation (Fig. 3h); thus, targeting COT kinase activity suppresses MEK/ERK phosphorylation in cancer cells with COT overexpression or amplification. Additionally, the targeting COT kinase activity in the presence of a B-RAF inhibitor (PLX4720) suppresses MEK/IRK phosphorylation (Fig. 3h). Treatment of RPMI-7951 cells with a small molecule COT kinase inhibitor (Wyeth, Abbot compound ID 9549300) (George, D. et al., Bioorg. Med. Chem. Lett. 18, 4952-4955 (2008); Hirata, K. et al., Biol. Pharm. Bull. 33, 133-137 (2010); Lee, K. M. et al, Cancer Res. 69, 8043-8049 (2009)) resulted in dose-dependent suppression of MEK and ERK phosphorylation, providing additional evidence that COT contributes to MEK/ERK activation in these cells (Fig. 3i).
[00123] Example 8: COT-expressing B-RAFV600E cell lines exhibit resistance to allosteric MEK inhibitors.
[00124] Whether COT-expressing cancer cells remain sensitive to MAPK pathway inhibition at a target downstream of COT or RAF was analyzed. The OUMS-23 and RPMI-7951 cell lines were queried for sensitivity to the MEK1/2 inhibitor CI-1040. Both cell lines were refractory to MEK inhibition (Fig. 4a) and displayed sustained ERK phosphorylation even at 1 μΜ CI-1040 (Fig. 4b). Ectopic COT expression in A375 and SKMEL28 cells also conferred decreased sensitivity to the MEK inhibitors CI-1040 and AZD6244, suggesting that COT expression alone was sufficient to induce this
phenotype (Figs. 4c, 4d and 21 ). Similar to results observed with pharmacological MEK inhibitors, MEK1/2 knockdown only modestly suppressed COT-mediated ERK phosphorylation in A375 cells (Fig. 22). These data demonstrate that COT activates ERK through MEK-independent and MEK-dependent mechanisms. Furthermore, an in vitro kinase assay using recombinant COT and ERK1 was performed, and it was demonstrated that recombinant COT induced pThr202/Tyr204 phosphorylation of ERK1 in vitro (Fig. 22). Thus, COT expression potentiates ERK activation in a
MEK-independent manner.
[00125] Example 9: Combinatorial MAPK pathway inhibition to suppress cell proliferation.
[00126] The use of RAF and MEK inhibitors in combination can override
resistance to single-agents as shown in Fig 23. It was tested whether the combined RAF/MEK inhibition might circumvent COT-driven resistance. In the setting of ectopic COT expression, exposure to AZD6244 or CI-1040 in combination with PLX470 (1 μΜ each) reduced cell growth and pERK expression more effectively than did single-agent PLX4720, even at concentrations of 10 μΜ (Figs. 4e, 4f and 23). These data underscore the importance of this pathway in B-RAFV600E tumor cells and demonstrate that dual B-RAF/MEK inhibition helps circumvent resistance to RAF inhibitors.
[00127] Methods
[00128] Center for Cancer Systems Biology (CCSB)/Broad Institute Kinase Open Reading Frame Collection
[00129] A library of 597 kinase ORFs in pDONR-223 Entry vectors (Invitrogen) was assembled. Individual clones were end-sequenced using vector-specific primers in both directions. Clones with substantial deviations from reported sequences were discarded. Entry clones and sequences are available via Addgene
(http://www.addgene.org/human_kinases). Kinase ORFs were assembled from multiple sources; 337 kinases were isolated as single clones from the ORFeome 5.1 collection (http://horfdb.dfci.harvard.edu), 183 kinases were cloned from normal human tissue RNA (Ambion) by reverse transcription and subsequent PCR amplification to add Gateway sequences (Invitrogen), 64 kinases were cloned from templates provided by the Harvard Institute of Proteomics (HIP), and 13 kinases were cloned into the Gateway system from templates obtained from collaborating laboratories. The Gateway- compatible lentiviral vector pLX-Blast-V5 was created from the pLKO.1 backbone. LR Clonase enzymatic recombination reactions were performed to introduce the 597 kinases into pLX-Blast-V5 according to the manufacturer's protocol (Invitrogen).
[00130] High throughput ORF screening
[00131] A375 melanoma cells were plated in 384-well microtiter plates (500 cells per well). The following day, cells were spin-infected with the lentivirally-packaged kinase ORF library in the presence of 8 ug/ml polybrene. 48 hours post-infection, media was replaced with standard growth media (2 replicates), media containing 1 μΜ
PLX4720 (2 replicates, 2 time points) or media containing 10 ug/ml blasticidin (2 replicates). After four days and 6 days, cell growth was assayed using Cell Titer-Glo (Promega) according to manufacturer instructions. The entire experiment was performed twice.
[00132] Identification of candidate resistance ORFs [00133] Raw luminescence values were imported into Microsoft Excel. Infection efficiency was determined by the percentage of duplicate-averaged raw
luminescence in blasticidin selected cells relative to non-selected cells. ORFs with an infection efficiency of less than 0.70 were excluded from further analysis along with any ORF having a standard deviation of >15,000 raw luminescence units between duplicates. To identify ORFs whose expression affects proliferation, we compared the duplicate-averaged raw luminescence of individual ORFs against the average and standard deviation of all control -treated cells via the z-score, or standard score, below,
χ - μ
Z =
σ where x = average raw luminescence of a given ORF, μ = the mean raw
luminescence of all ORFs and σ = the standard deviation of the raw luminescence of all wells. Any individual ORF with a z-score >+2 or <-2 was annotated as affecting proliferation and removed from final analysis. Differential proliferation was
determined by the percentage of duplicate-averaged raw luminescence values in PLX4720 (1 μΜ) treated cells relative to untreated cells. Subsequently, differential proliferation was normalized to the positive control for PLX4720 resistance,
MEK1S218 222D (MEK1 DD), with MEK1 DD differential proliferation = 1.0. MEK1 DD normalized differential proliferation for each individual ORF was averaged across two duplicate experiments, with two time points for each experiment (day 4 and day 6). A z-score was then generated, as described above for average MEK1 DD normalized differential proliferation. ORFs with a z-score of >2 were considered hits and were followed up in the secondary screen.
[00134] ORF and shRNA expression
[00135] ORFs were expressed from pLX-Blast-V5 (lentiviral) or pWZL-Blast, pBABE-Puro or pBABE-zeocin (retroviral) expression plasmids. For lentiviral transduction, 293T cells were transfected with 1 μg of pLX-Blast-V5-ORF or pLKO. I -shRNA, 900 ng Δ8.9 (gag, pol) and 100 ng VSV-G using 6 μΙ Fugene6 transfection reagent (Roche). Viral supernatant was harvested 72 h
post-transfection. Mammalian cells were infected at a 1 : 10-1 :20 dilution of virus in 6-well plates in the presence of 5 μg/ml polybrene and centrifuged at 2250 RPM for 1 h at 37°C. Twenty-four hours after infection blasticidin (pLX-Blast-V5, 10 μg/ml) or puro (pLKO.1 , 0.75 μg/ml) was added and cells were selected for 48 hrs. For retrovirus production, 293T were transfected with 1 μg of retroviral plasmid-ORF, 1 μg pCL-AMPHO and 100 ng VSV-G, as described above. Cells were infected with retrovirus containing supernatant at a 1 :2 dilution in 5 μg/ml polybrene overnight, followed by media change to growth medium. Infection was repeated once more (twice total), followed by selection, above.
[00136] Secondary screen
[00137] A375 (1 .5 x 103) and SKMEL28 cells (3 x 103) were seeded in 96-well plates for 18 h. ORF-expressing lentivirus was added at a 1 : 10 dilution in the presence of 8 μg/ml polybrene, and centrifuged at 2250 RPM and 37° C for 1 h. Following centrifugation, virus-containing media was changed to normal growth media and allowed to incubate for 18 h. Twenty-four hours after infection, DMSO (1 : 1000) or 10x PLX4720 (in DMSO) was added to a final concentration of 100, 10, 1 , 0.1 , 0.01 , 0.001 , 0.0001 or 0.00001 μΜ. Cell viability was assayed using WST-1 (Roche), per manufacturer recommendation, 4 days after the addition of PLX4720.
[00138] Cell lines and reagents
[00139] A375, SKMEL28, SKMEL30, COLO-679, WM451 lu, SKMEL5, Malme 3M, SKMEL30, WM3627, WM1976, WM3163, WM3130, WM3629, WM3453, WM3682 and WM3702 were all grown in RPMI (Cellgro), 10% FBS and 1 % penicillin/streptomycin. M307 was grown in RPMI (Cellgro), 10% FBS and 1 % penicillin/streptomycin supplemented with 1 mM sodium pyruvate. 293T and
OUMS-23 were grown in DMEM (Cellgro), 10% FBS and 1 % penicillin/streptomycin. RPMI-7951 cells (ATCC) were grown in MEM (Cellgro), 10% FBS and 1 %
penicillin/streptomycin. Wild-type primary melanocytes were grown in HAM's F10 (Cellgro), 10% FBS and 1 % penicillin/streptomycin. B-RAFV600E-expressing primary melanocytes were grown in TIVA media [Ham's F-10 (Cellgro), 7% FBS, 1 % penicillin/streptomycin, 2mM glutamine (Cellgro), 100 uM IBMX, 50 ng/ml TPA, 1 mM dbcAMP (Sigma) and 1 μΜ sodium vanadate]. CI-1040 (PubChem ID: 6918454) was purchased from Shanghai Lechen International Trading Co., AZD6244
(PubChem ID: 10127622) from Selleck Chemicals, and PLX4720 (PubChem ID: 24180719) from Symansis. RAF265 (PubChem ID: 1 1656518) was a generous gift from Novartis Pharma AG. Unless otherwise indicated, all drug treatments were for 16 h. Activated alleles of NRAS and KRAS have been previously described.
(Boehm, J. S. et al. Ceil 129, 1065-1079 (2007); Lundberg, A. S. et al. Oncogene 21 , 4577-4586 (2002)).
[00140] Pharmacologic growth inhibition assays
[00141] Cultured cells were seeded into 96-well plates (3,000 cells per well) for all melanoma cell lines; 1 ,500 cells were seeded for A375. Twenty-four hours after seeding, serial dilutions of the relevant compound were prepared in DMSO added to cells, yielding final drug concentrations ranging from 100 μΜ to 1 x 105 μΜ, with the final volume of DMSO not exceeding 1 %. Cells were incubated for 96 h following addition of drug. Cell viability was measured using the WST1 viability assay (Roche). Viability was calculated as a percentage of control (untreated cells) after background subtraction. A minimum of six replicates were performed for each cell line and drug combination. Data from growth-inhibition assays were modeled using a nonlinear regression curve fit with a sigmoid dose-response. These curves were displayed and GI50 generated using GraphPad Prism 5 for Windows (GraphPad). Sigmoid-response curves that crossed the 50% inhibition point at or above 10 μΜ have GI50 values annotated as >10 μΜ. For single-dose studies, the identical protocol was followed, using a single dose of indicated drug (1 μΜ unless otherwise noted).
[00142] Immunoblots and immunoprecipitations
[00143] Cells were washed twice with ice-cold PBS and lysed with 1 % NP-40 buffer [150 mM NaCI, 50 mM Tris pH 7.5, 2 mM EDTA pH 8, 25 mM NaF and 1 % NP-40] containing 2x protease inhibitors (Roche) and 1x Phosphatase Inhibitor Cocktails I and II (CalBioChem). Lysates were quantified (Bradford assay), normalized, reduced, denatured (95°C) and resolved by SDS gel electrophoresis on 10% Tris/Glycine gels (Invitrogen). Protein was transferred to PVDF membranes and probed with primary antibodies recognizing pERK1/2 (T202/Y204), pMEK1/2 (S217/221 ), MEK1/2, MEK1 , MEK2, C-RAF (rabbit host), pC-RAF (pS338) (Cell Signaling Technology; 1 : 1 ,000), V5-HRP (Invitrogen; (1 :5,000), COT (1 :500), B-RAF (1 :2,000), Actin (1 :1 ,000), Actin-HRP (1 :1 ,000; Santa Cruz)), C-RAF (mouse host; 1 :1 ,000; BD Transduction Labs), Vinculin (Sigma; 1 :20,000), AXL (1 :500; R&D Systems). After incubation with the appropriate secondary antibody (anti-rabbit, anti-mouse IgG, HRP-linked; 1 :1 ,000 dilution, Cell Signaling Technology or anti-goat IgG, HRP-linked; 1 : 1 ,000 dilution; Santa Cruz), proteins were detected using chemiluminescence (Pierce). Immunoprecipitations were performed overnight at 4° C in 1 % NP-40 lysis buffer, as described above, at a concentration of 1 μ9/μΙ total protein using an antibody recognizing C-RAF (1 :50; Cell Signaling Technology). Antibody: antigen complexes were bound to Protein A agarose (25 μΙ_, 50% slurry; Pierce) for 2 hrs. at 4° C. Beads were centrifuged and washed three times in lysis buffer and eluted and denatured (95 °C) in 2x reduced sample buffer (Invitrogen). Immunoblots were performed as above. Phospho-protein quantification was performed using NIH Image J.
[00144] Lysates from tumor and matched normal skin were generated by mechanical homogenization of tissue in RIPA [50 mM Tris (pH 7.4), 150 mM NaCI, 1 mM EDTA, 0.1 % SDS, 1 .0% NaDOC, 1 .0% Triton X-100, 25 mM NaF,
1 mM NA3VO4] containing protease and phosphatase inhibitors, as above.
Subsequent normalization and immunoblots were performed as above.
[00145] Biopsied melanoma tumour material
[00146] Biopsied tumor material consisted of discarded and de-identified tissue that was obtained with informed consent and characterized under protocol 02-017 (paired samples, Massachusetts General Hospital) and 07-087 (unpaired sample, Dana-Farber Cancer Institute). For paired specimens, Όη-treatment' samples were collected 10-14 days after initiation of PLX4032 treatment (Table 6).
[00147] Inhibition of COT kinase activity [00148] Adherent RPMI-7951 cells were washed twice with 1x PBS and incubated overnight in serum-free growth media. Subsequently,
4-(3-Chloro-4-fluorophenylamino)-6-(pyridin-3-yl-methylamino)-3-cyano-[1 ,7]- naphthyridine (EMD;TPL2 inhibitor I; Cat#: 616373, PubChem ID: 9549300), suspended in DMSO at the indicated concentration, was added to cells for 1 hour, after which protein extracts were made as described above.
[00149] Quantitative RT-PCR
[00150] mRNA was extracted from cell lines and fresh-frozen tumors using the RNeasy kit (Qiagen). Total mRNA was used for subsequent reverse transcription using the Superscript III First-Strand Synthesis SuperMix (Invitrogen) for cell lines and unpaired tumor samples, and the Superscript VILO cDNA synthesis kit
(Invitrogen) for paired frozen tumor samples. 5 μΙ of the RT reaction was used for quantitative PCR using SYBR Green PCR Master Mix and gene-specific primers, in triplicate, using an ABI 7300 Real Time PCR System. Primers used for detection are as follows:
[00151] In vitro kinase assay
[00152] In vitro kinase assays were performed as previously described using 1 μg each of COT (amino acids 30- 397, R&D Systems) and inactive ERK1 (Millipore).
[00153] Cellular viability assays
[00154] Adherent RPMI-7951 cells were infected with virus expressing shRNAs against COT or Luciferase as described above. Following selection, cells were plated (1 .5x105 cells/well) onto a 24-well plate in quadruplicate. Viable cells were counted via trypan blue exclusion using a VI-CELL Cell Viability Analyzer, per manufacturer's specifications. Quadruplicate cell counts were averaged and normalized relative to that of the control shRNA.
[00155] The Cancer Cell Line Encyclopedia (CCLE)
[00156] The Cancer Cell Line Encyclopedia (CCLE) project is a collaboration between the Broad Institute, the Novartis Institutes for Biomedical Research (NIBR) and the Genomics Institute of the Novartis Research Foundation (GNF) to conduct a detailed genetic and pharmacologic characterization of a large panel of human cancer models, to develop integrated computational analyses that link distinct pharmacologic vulnerabilities to genomic patterns and to translate cell line integrative genomics into cancer patient stratification. Chromosomal copy number and gene expression data used for this study are available online at http://www.broadinstitute.org/cgi- bin/cancer/datasets.cgi.
[00157] Expression profiling of cancer cell lines
[00158] Oligonucleotide microarray analysis was carried out using the GeneChip Human Genome U133 Plus 2.0 Affymetrix expression array (Affymetrix). Samples were converted to labeled, fragmented, cRNA following the Affymetrix protocol for use on the expression microarray.
[00159] shRNA constructs used (pLKO.1 )
[00160] The DNA sequences for preparing the shRNA constructs used were as follows:
0000001066 1236s1 c1 NO. 10 shCRAF(2) TRCN NM_002880.x- GAGACATGAAATCCAACAATA SEQ. ID.
0000001068 1529s1 c1 NO. 1 1 shMEK1 (1 ) TRCN NM_002755.x- GATTACATAGTCAACGAGCCT SEQ. ID.
0000002332 1015s1 c1 NO. 12 shMEK1 (2) TRCN NM_002755.x- G CTTCTATG GTGC GTTCTACA SEQ. ID.
0000002329 455s1 c1 NO. 13 shMEK2(1 ) TRCN NM_030662.2- TGGACTATATTGTGAACGAGC SEQ. ID.
0000007007 1219s1 c1 NO. 14 shMEK2(2) TRCN NM_030662.2- CCAACATCCTCGTGAACTCTA SEQ. ID.
0000007005 847s1 c1 NO. 15 shCOT(1 ) TRCN NM_005204.x- C AAG AG C C G C AG AC CTACTAA SEQ. ID.
0000010013 1826s1 c1 NO. 16 shCOT(2) TRCN NM_005204.2- GATGAGAATGTGACCTTTAAG SEQ. ID.
0000196518 2809s1 c1 NO. 17
[00161] The definitions and disclosures provided herein govern and supersede all others incorporated by reference. Although the invention herein has been described in connection with preferred embodiments thereof, it will be appreciated by those skilled in the art that additions, modifications, substitutions, and deletions not specifically described may be made without departing from the spirit and scope of the invention as defined in the appended claims. It is therefore intended that the foregoing detailed description be regarded as illustrative rather than limiting, and that it be understood that it is the following claims, including all equivalents, that are intended to define the spirit and scope of this invention.
Table 3: CCSB/Broad Institute Kinase ORF Library description and ORF classification Abbreviations: RS/TK (Receptor Serine/Threonine Kinase); RTK (Receptor Tyrosine Kinase); NRS/TK(Non-Receptor Serine/Threonine Kinase); NRTK (Non-
proen nase reguaory su un proen nase
sco n oman recepor am y, mem er proen nase
ERBB4 2066 v-erb-a erythroblastic leukemia viral oncogene homolog 4 (avian) protein kinase
05 3);unassigned (NRS/TK)
m ogen-ac vae proen nase nase proen nase
PLXNA3 55558 plexin A3;PLXNA3 protein kinase (RTK)
nase yeas proen nase
TSSK6 83983 serine/threonine protein kinase SSTK protein kinase
Table 4: Ranking of average differential proliferation (1 μΜ PLX4720/control) for 597 kinase-
RANK GENE AVERAGE STDEV. Z-SCORE NOTES
39 LOC340156 50.59% 14.63% 0.88
40 MAP4K5 50.57% 35.45% 0.88
41 ABL1 50.55% 19.09% 0.87
42 MAP2K1IP1 50.47% 11.54% 0.86
43 PRKAR1B 50.34% 8.31 % 0.84
44 RPS6KA1 50.12% 12.28% 0.81
45 TSSK6 50.10% 11.70% 0.81
46 NEK9 50.04% 14.37% 0.80
47 DLG1 49.98% 12.07% 0.80
48 PTK6 49.93% 10.93% 0.79
49 SCYL2 49.85% 12.51% 0.78
50 STK11 49.82% 12.10% 0.77
51 C9orf98 49.82% 10.38% 0.77
52 PCK2 49.77% 12.93% 0.77
53 NTRK2 49.77% 10.02% 0.77
54 TRIM27 49.69% 10.38% 0.75
55 FN3KRP 49.69% 11.83% 0.75
56 CAMK2D 49.61 % 11.93% 0.74
57 ALPK2 49.56% 10.41% 0.74
58 LOC652722 49.52% 13.10% 0.73
59 LATS2 49.49% 12.45% 0.73
60 STK3 49.48% 10.15% 0.73
61 CDC42BPG 49.44% 10.19% 0.72
62 HKDC1 49.44% 11.53% 0.72
63 CDK7 49.42% 14.45% 0.72 Proliferative
64 WNK4 49.37% 14.07% 0.71
65 PRPF4B 49.36% 12.18% 0.71
66 MAPKAPK2 49.30% 12.83% 0.70
67 MEK WT 49.29% 14.33% 0.70 (-) Control
68 PRPS1 49.28% 12.09% 0.70
69 BTK 49.22% 14.59% 0.69
70 MY03B 49.19% 11.05% 0.69
71 TESK1 49.18% 12.19% 0.68
72 PLXNA3 49.11 % 9.04% 0.67
73 CLK2 49.09% 8.01 % 0.67
74 PFKFB1 49.02% 14.04% 0.66
75 DAK 49.00% 12.68% 0.66
76 ITPKB 48.99% 16.62% 0.66
77 CHEK1 48.98% 14.41% 0.66
78 MERTK 48.94% 12.88% 0.65
79 IKBKE 48.83% 12.46% 0.63
80 CARD11 48.83% 15.02% 0.63 RANK GENE AVERAGE STDEV. Z-SCORE NOTES
81 PANK3 48.78% 9.70% 0.63
82 TRIB2 48.76% 10.71% 0.63
83 CDC7 48.74% 9.83% 0.62
84 PIK3C3 48.69% 12.05% 0.62
85 LRGUK 48.60% 11.29% 0.60
86 SCYL3 48.58% 10.86% 0.60
87 MAP3K14 48.57% 8.10% 0.60
88 LOC730000 48.57% 13.36% 0.60
89 LOC442075 48.55% 10.75% 0.60
90 LOC54103 48.50% 10.77% 0.59
91 NPR2 48.49% 13.29% 0.59
92 COL4A3BP 48.49% 11.10% 0.59
93 MYLK2 48.49% 11.83% 0.59
94 CSNK2B 48.48% 11.52% 0.59
95 CARKL 48.47% 9.19% 0.58
96 TP53RK 48.44% 14.25% 0.58 Proliferative
97 PRKCG 48.42% 8.31 % 0.58
98 ALK 48.39% 14.07% 0.57
99 ETNK1 48.37% 11.25% 0.57
100 DYRK2 48.36% 11.80% 0.57
101 DGKA 48.31 % 10.71% 0.56
102 ADPGK 48.30% 13.36% 0.56
103 PRKAB2 48.29% 10.75% 0.56
104 ULK3 48.28% 11.18% 0.56
105 KIAA2002 48.28% 12.39% 0.56
106 IRAK3 48.26% 10.96% 0.56
107 TYK2 48.24% 10.88% 0.55
108 MAP2K7 48.23% 12.06% 0.55
109 NRBP 48.18% 13.40% 0.54
110 CDK2 48.14% 14.65% 0.54
111 MORN2 47.98% 11.36% 0.52
112 EPHB4 47.92% 12.52% 0.51
113 PRKCZ 47.88% 12.68% 0.50
114 RFK 47.85% 10.44% 0.50
115 RAGE 47.83% 10.46% 0.50
116 LOC91807 47.80% 11.25% 0.49
117 PIK3CG 47.76% 11.25% 0.49
118 PIK3C2G 47.74% 10.75% 0.48
119 ARSG 47.71 % 10.26% 0.48
120 CSNK1A1L 47.66% 10.26% 0.47
121 ALS2CR7 47.65% 11.56% 0.47
122 FLJ40852 47.62% 12.34% 0.47 RANK GENE AVERAGE STDEV. Z-SCORE NOTES
123 LOC390877 47.61 % 11.43% 0.46
124 TRIB1 47.59% 12.91% 0.46
125 TPR 47.58% 13.00% 0.46
126 RPS6KL1 47.57% 12.33% 0.46
127 CCRK 47.56% 10.28% 0.46
128 PDPK1 47.53% 10.42% 0.45
129 FASTKD5 47.52% 10.70% 0.45
130 CDKL3 47.49% 13.14% 0.45
131 DTYMK 47.49% 10.60% 0.45
132 MPP3 47.47% 10.65% 0.45
133 HSPB8 47.46% 11.73% 0.44
134 NME6 47.44% 14.48% 0.44
135 NEK3 47.42% 15.46% 0.44
136 PIK3R4 47.41 % 11.14% 0.44
137 MGC16169 47.41 % 13.90% 0.44
138 OXSR1 47.40% 11.68% 0.44
139 MASTL 47.35% 10.28% 0.43
140 PNCK 47.35% 10.15% 0.43
141 ADCK2 47.32% 15.57% 0.43
142 SNX16 47.26% 12.76% 0.42
143 HCK 47.24% 23.41% 0.41
144 CDKL2 47.21 % 10.60% 0.41
145 NEK11 47.20% 14.68% 0.41
146 BMP2K 47.15% 11.18% 0.40
147 BUB1 47.15% 11.76% 0.40
148 PINK1 47.13% 11.38% 0.40
149 RBKS 47.08% 8.73% 0.39
150 LOC732306 47.03% 8.82% 0.39
151 PKLR 47.03% 12.10% 0.38
152 DYRK1A 47.01 % 12.69% 0.38
153 RPS6KA4 47.01 % 11.70% 0.38
154 DGKG 46.94% 12.71% 0.37
155 CDK5R1 46.93% 9.40% 0.37
156 FLJ25006 46.87% 11.64% 0.36
157 PBK 46.86% 14.05% 0.36
158 ACVR1C 46.82% 9.98% 0.35
159 FLT1 46.78% 10.42% 0.35
160 PLXNA4B 46.76% 9.39% 0.35
161 MVK 46.71 % 9.26% 0.34
162 LIMK2 46.70% 11.29% 0.34
163 DYRK1B 46.68% 20.75% 0.34
164 MARK3 46.67% 11.71% 0.34 Proliferative RANK GENE AVERAGE STDEV. Z-SCORE NOTES
165 BMPR1B 46.66% 12.24% 0.33
166 NUP62 46.63% 9.92% 0.33
167 JAK2 46.62% 12.50% 0.33
168 MAPK12 46.55% 9.98% 0.32
169 DDR2 46.54% 7.99% 0.32
170 MAK 46.52% 15.42% 0.31
171 GLYCTK 46.50% 13.19% 0.31
172 AK1 46.47% 9.68% 0.31
173 MAPK15 46.47% 11.14% 0.31
174 MAST1 46.45% 10.77% 0.30
175 PAPSS2 46.39% 11.53% 0.30
176 CSF1R 46.34% 12.86% 0.29
177 TPK1 46.29% 9.52% 0.28
178 PAK4 46.24% 9.98% 0.28
179 NAGK 46.21 % 12.34% 0.27
180 CDK8 46.20% 7.68% 0.27
181 STK40 46.19% 12.92% 0.27
182 CIB1 46.10% 10.92% 0.26
183 PLK1 46.08% 9.99% 0.25
184 FLJ23356 46.04% 10.45% 0.25
185 LOC220686 46.04% 9.31 % 0.25
186 PRKAB1 46.03% 12.00% 0.25
187 JAK3 46.02% 10.17% 0.24
188 NME1 46.02% 13.01% 0.24
189 NME5 46.00% 11.53% 0.24
190 PER 45.97% 9.92% 0.24
191 AK3L1 45.97% 10.39% 0.24
192 PRKG2 45.90% 13.24% 0.23
193 RP6-213H19.1 45.88% 12.83% 0.22
194 AKT3 45.87% 11.32% 0.22
195 PSKH1 45.86% 10.16% 0.22
196 PRKAR2B 45.84% 13.05% 0.22
197 FUK 45.81 % 10.94% 0.22
198 ADCK4 45.79% 11.25% 0.21
199 TEC 45.78% 11.46% 0.21
200 PRKAG1 45.77% 11.08% 0.21
201 FXN 45.77% 12.03% 0.21
202 AAK1 45.68% 11.49% 0.20
203 CAMK2B 45.67% 19.84% 0.20
204 COASY 45.64% 11.07% 0.19
205 PRKAG3 45.63% 10.21% 0.19
206 NME7 45.58% 10.18% 0.18 RANK GENE AVERAGE STDEV. Z-SCORE NOTES
207 LMTK2 45.58% 12.15% 0.18
208 PANK2 45.57% 11.72% 0.18
209 PRKD3 45.47% 13.14% 0.17
210 PHKA2 45.47% 10.92% 0.17
211 SLAMF6 45.46% 9.67% 0.17
212 SRPK1 45.46% 12.54% 0.17
213 HIPK1 45.42% 12.59% 0.16
214 EPHA1 45.39% 10.59% 0.16
215 WNK1 45.38% 13.79% 0.15
216 PDIK1L 45.33% 12.31% 0.15
217 BMP2KL 45.33% 10.08% 0.15
218 MAP3K5 45.32% 14.13% 0.15
219 EPHA2 45.30% 12.87% 0.14
220 CCL2 45.29% 9.71 % 0.14
221 CDKL1 45.25% 10.13% 0.14
222 NJMU-R1 45.23% 8.13% 0.13
223 LOC652799 45.18% 10.30% 0.13
224 GUK1 45.15% 12.23% 0.12
225 NME4 45.12% 13.03% 0.12
226 YSK4 45.11 % 13.47% 0.12
227 NEK2 45.11 % 9.38% 0.12
228 C9orf95 45.10% 9.38% 0.12
229 CDC2 45.05% 11.95% 0.11
230 FGFR2 45.01 % 11.23% 0.10
231 IPMK 44.98% 10.67% 0.10
232 STK32C 44.98% 12.10% 0.10
233 PIP5K2A 44.92% 14.55% 0.09
234 PRKX 44.90% 10.44% 0.09
235 TRPM7 44.80% 10.36% 0.07
236 FLJ10986 44.74% 10.95% 0.07
237 SNF1LK 44.74% 12.70% 0.07
238 MAP3K6 44.71 % 9.75% 0.06
239 LOC653052 44.70% 14.56% 0.06
240 IRAK2 44.69% 11.61% 0.06
241 XYLB 44.68% 11.47% 0.06
242 PTK7 44.67% 14.55% 0.06
243 PKMYT1 44.66% 13.74% 0.05
244 SPHK2 44.63% 10.35% 0.05
245 NME2 44.63% 11.57% 0.05
246 PRKAA1 44.62% 15.03% 0.05
247 MAPK14 44.60% 10.77% 0.05
248 NTRK3 44.58% 13.43% 0.04 RANK GENE AVERAGE STDEV. Z-SCORE NOTES
249 PRKACB 44.58% 9.30% 0.04
250 LOC650122 44.56% 11.24% 0.04
251 CDK6 44.51 % 10.74% 0.03
252 AMHR2 44.48% 11.11% 0.03
253 IPPK 44.42% 10.63% 0.02
254 AK7 44.41 % 10.36% 0.02
255 PIP5KL1 44.36% 11.74% 0.01
256 LOC340371 44.30% 16.04% 0.01
257 DKFZp434B1231 44.29% 12.21% 0.00
258 PRKAA2 44.28% 11.42% 0.00
259 FASTKD1 44.28% 10.73% 0.00
260 ARAF 44.27% 14.40% 0.00 RAF Family
261 HK3 44.26% 14.83% 0.00
262 KSR2 44.25% 10.28% 0.00
263 PAK7 44.24% 18.45% 0.00
264 GTF2H1 44.23% 10.90% 0.00
265 RPS6KB2 44.23% 9.32% -0.01
266 PAK6 44.21 % 13.83% -0.01
267 IRAK4 44.18% 10.27% -0.01
268 NLK 44.18% 12.92% -0.01
269 FYN 44.14% 12.43% -0.02
270 BMPR2 44.14% 12.20% -0.02
271 CDK4 44.13% 10.47% -0.02
272 STK32A 44.07% 13.93% -0.03
273 TNK1 44.06% 10.80% -0.03
274 STK24 43.99% 10.79% -0.04
275 CSNK2A1 43.99% 11.97% -0.04
276 AK3 43.98% 11.16% -0.04
277 TTK 43.93% 10.47% -0.05
278 PGK2 43.91 % 13.31% -0.05
279 GALK1 43.89% 10.14% -0.05
280 DYRK3 43.89% 9.32% -0.05
281 EIF2AK4 43.83% 11.00% -0.06
282 PDK1 43.79% 13.31% -0.07
283 EIF2AK1 43.79% 12.02% -0.07
284 PRKACG 43.79% 11.37% -0.07
285 BUB1B 43.74% 10.69% -0.07
286 PKM2 43.74% 14.46% -0.07
287 LRPPRC 43.64% 10.87% -0.09
288 EPHA6 43.63% 12.60% -0.09
289 HK2 43.58% 12.55% -0.10
290 MET 43.55% 10.99% -0.10 RANK GENE AVERAGE STDEV. Z-SCORE NOTES
291 CKS2 43.53% 7.83% -0.10
292 LOC375133 43.53% 10.05% -0.10
293 LIMK1 43.52% 12.43% -0.10
294 TWF2 43.50% 11.71% -0.11
295 PCTK2 43.47% 12.02% -0.11
296 DCAKD 43.43% 11.61% -0.12
297 PLXNB2 43.41 % 11.85% -0.12
298 MST1R 43.35% 11.99% -0.13
299 PRKAR1A 43.35% 9.03% -0.13
300 SEPHS2 43.32% 9.68% -0.13
301 SPHK1 43.31 % 9.87% -0.13
302 CCL4 43.30% 11.83% -0.13
303 KIAA0999 43.28% 9.68% -0.14
304 PDGFRL 43.23% 11.93% -0.14
305 BRSK1 43.21 % 14.17% -0.15
306 BRAF 43.21 % 12.56% -0.15 RAF Family
307 MUSK 43.20% 11.03% -0.15
308 TNNI3K 43.20% 10.46% -0.15
309 GK2 43.18% 15.15% -0.15
310 CKB 43.14% 9.45% -0.16
311 DGKB 43.12% 11.62% -0.16
312 LOC648152 42.99% 11.15% -0.18
313 RPS6KA5 42.87% 11.23% -0.19
314 CASK 42.87% 12.05% -0.19
315 PHKA1 42.86% 10.10% -0.20
316 AK2 42.84% 9.83% -0.20
317 PIM1 42.81 % 14.09% -0.20
318 ZAP70 42.81 % 12.10% -0.20
319 PNKP 42.79% 12.95% -0.20
320 CDK10 42.78% 9.77% -0.21
321 CHEK2 42.77% 12.86% -0.21
322 CAMK2A 42.72% 11.68% -0.21
323 CAMK2G 42.70% 9.66% -0.22
324 ADRBK2 42.70% 12.56% -0.22
325 NEK8 42.69% 9.88% -0.22
326 PRKR 42.69% 10.65% -0.22
327 CHKA 42.66% 12.84% -0.22
328 ACVRL1 42.65% 10.85% -0.22
329 PRKY 42.63% 11.80% -0.23
330 TRIB3 42.62% 11.45% -0.23
331 PRKD2 42.59% 10.48% -0.23
332 PIP5K3 42.57% 9.78% -0.24 RANK GENE AVERAGE STDEV. Z-SCORE NOTES
333 LOC727761 42.56% 14.17% -0.24
334 PTK2B 42.50% 15.72% -0.25
335 MPP2 42.48% 10.73% -0.25
336 CSNK1A1 42.47% 13.54% -0.25
337 PTK9 42.42% 11.31% -0.26
338 STK25 42.41 % 12.54% -0.26
339 PFKL 42.39% 12.01% -0.26
340 STK19 42.35% 11.20% -0.27
341 PI4KII 42.30% 14.04% -0.27
342 HK1 42.26% 12.73% -0.28
343 AURKA 42.21 % 14.80% -0.29
344 GK5 42.21 % 10.98% -0.29
345 MAP3K7 42.20% 13.13% -0.29
346 PFTK1 42.18% 12.49% -0.29
347 ERN1 42.17% 13.74% -0.29
348 STK33 42.17% 13.22% -0.29
349 SYK 42.13% 12.42% -0.30
350 GALK2 42.13% 11.34% -0.30
351 TSSK1 42.12% 11.98% -0.30
352 MAPK6 42.11 % 10.58% -0.30
353 ASCII 42.07% 10.61% -0.30
354 PFKP 42.07% 14.99% -0.31
355 PXK 42.05% 14.69% -0.31
356 PI4K2B 42.05% 11.84% -0.31
357 PIP5K2C 42.02% 9.50% -0.31
358 PFKFB3 42.00% 12.79% -0.31
359 TSSK3 41.97% 14.82% -0.32
360 MPP6 41.97% 12.68% -0.32
361 MPP4 41.87% 12.27% -0.33
362 LOC653155 41.85% 11.50% -0.34
363 ALPK1 41.82% 12.37% -0.34
364 CDK9 41.76% 10.32% -0.35
365 PDK3 41.66% 10.87% -0.36
366 CKMT2 41.66% 12.57% -0.36
367 CAMK1G 41.63% 12.06% -0.37
368 MAPKAPK3 41.61 % 13.19% -0.37
369 PIK4CB 41.55% 14.29% -0.38
370 PRPS1L1 41.54% 11.08% -0.38
371 FASTK 41.49% 12.56% -0.39
372 CAMK1D 41.49% 11.54% -0.39
373 MARK2 41.48% 14.28% -0.39
374 PDK4 41.43% 13.43% -0.39 RANK GENE AVERAGE STDEV. Z-SCORE NOTES
375 NEK7 41.36% 9.57% -0.40
376 MAPK4 41.34% 10.83% -0.41
377 PIK4CA 41.32% 11.99% -0.41
378 JAK1 41.31 % 11.56% -0.41
379 PDXK 41.30% 11.93% -0.41
380 TGFBR2 41.25% 15.85% -0.42
381 PHKB 41.25% 10.81% -0.42
382 ULK2 41.24% 15.35% -0.42
383 MKNK1 41.21 % 13.08% -0.42
384 CDC2L6 41.17% 10.91% -0.43
385 CSNK1G3 41.13% 10.53% -0.44
386 CAMK1 41.08% 11.68% -0.44
387 DGKZ 41.07% 12.69% -0.44
388 SGK 40.91 % 11.92% -0.47
389 TBK1 40.83% 14.81% -0.48
390 ILK 40.81 % 12.62% -0.48
391 STK32B 40.80% 11.79% -0.48
392 TXNDC3 40.78% 12.24% -0.48
393 RPS6KB1 40.72% 10.13% -0.49
394 ZAK 40.71 % 10.32% -0.49
395 DYRK4 40.66% 12.59% -0.50
396 ITGB1BP3 40.57% 11.99% -0.51
397 MPP1 40.53% 10.94% -0.52
398 HIPK2 40.49% 11.06% -0.52
399 MAPK13 40.46% 12.06% -0.53
400 TK1 40.39% 11.04% -0.54
401 SH3BP4 40.36% 12.11% -0.54
402 CKM 40.29% 12.70% -0.55
403 FGFRL1 40.23% 16.94% -0.56
404 MAPK10 40.23% 12.29% -0.56
405 CALM2 40.16% 8.64% -0.57
406 CALM3 40.12% 11.85% -0.58
407 STYK1 40.09% 11.47% -0.58
408 CDKL4 40.08% 11.01% -0.58
409 NADK 40.08% 11.89% -0.58
410 MPP7 40.06% 11.72% -0.59
411 CAMKV 40.03% 12.16% -0.59
412 EXOSC10 40.02% 12.78% -0.59
413 CDKL5 39.98% 10.10% -0.60
414 STK38 39.90% 9.61 % -0.61
415 INSRR 39.90% 15.08% -0.61
416 DCK 39.86% 10.23% -0.61 RANK GENE AVERAGE STDEV. Z-SCORE NOTES
417 TLK2 39.80% 14.66% -0.62
418 PCTK3 39.66% 10.48% -0.64
419 LOC388957 39.65% 10.97% -0.64
420 PAPSS1 39.59% 13.48% -0.65
421 ACVR2B 39.56% 14.70% -0.65
422 NME1-NME2 39.53% 12.01% -0.66
423 NEK10 39.50% 13.78% -0.66
424 MAP3K11 39.47% 9.33% -0.67
425 PMVK 39.46% 10.19% -0.67
426 MAPK9 39.45% 12.74% -0.67
427 MKNK2 39.39% 13.54% -0.68
428 GRK7 39.34% 14.98% -0.68
429 RIOK2 39.27% 11.79% -0.69
430 DGKK 39.27% 9.02% -0.69
431 ACVR1 39.15% 13.96% -0.71
432 TLK1 39.11 % 15.46% -0.72
433 LATS1 39.05% 10.46% -0.73
434 SCYL1 39.01 % 11.41% -0.73
435 TESK2 38.93% 13.30% -0.74
436 DGUOK 38.90% 14.54% -0.75
437 PGK1 38.87% 12.26% -0.75
438 MAGI1 38.87% 12.66% -0.75
439 SNRK 38.79% 11.61% -0.76
440 CALM1 38.65% 10.26% -0.78
441 RIOK1 38.58% 12.39% -0.79
442 EEF2K 38.56% 11.96% -0.79
443 MAPK8 38.45% 10.51% -0.81
444 CSNK1D 38.44% 15.60% -0.81
445 ULK4 38.42% 12.02% -0.81
446 STK38L 38.33% 12.63% -0.83
447 RIOK3 38.21 % 12.80% -0.84
448 MINK1 38.00% 14.94% -0.87
449 ROR2 37.95% 15.32% -0.88
450 PTK2 37.93% 10.59% -0.88
451 PIK3R5 37.84% 13.17% -0.89
452 ALDH18A1 37.81 % 15.66% -0.90
453 NYD-SP25 37.79% 13.52% -0.90
454 MAP4K3 37.76% 9.58% -0.90
455 AGK 37.61 % 13.89% -0.92
456 GSK3A 37.56% 14.44% -0.93
457 BMPR1A 37.56% 16.10% -0.93
458 STK16 37.53% 11.09% -0.94 RANK GENE AVERAGE STDEV. Z-SCORE NOTES
459 FASTKD2 37.50% 8.53% -0.94
460 MAP4K4 37.39% 15.68% -0.96
461 LRRK2 37.38% 10.11% -0.96
462 TGFBR3 37.33% 11.04% -0.96
463 PDGFRB 37.29% 16.90% -0.97
464 DLG3 37.09% 12.08% -1.00
465 PFKFB2 36.99% 10.37% -1.01
466 AKT1 36.66% 13.43% -1.06
467 PRKCI 36.54% 11.44% -1.07
468 NEK4 36.50% 13.19% -1.08
469 PRKD1 36.45% 14.57% -1.09
470 SRPK2 36.37% 12.38% -1.10
471 SH3BP5 36.37% 17.17% -1.10
472 CLK1 36.06% 11.13% -1.14
473 GK 35.86% 17.33% -1.17
474 IHPK1 35.53% 12.15% -1.21
475 IHPK3 35.48% 9.30% -1.22
476 PLAU 35.36% 13.10% -1.24
477 TAOK3 35.29% 14.78% -1.25
478 PAK2 35.20% 12.51% -1.26
479 BMX 35.12% 12.94% -1.27
480 DAPK2 35.01% 13.92% -1.29
481 CSNK1E 34.83% 15.21% -1.31
482 MAPK3 34.75% 19.93% -1.32 Lethal
483 MAP3K15 34.40% 16.76% -1.37 Lethal
484 MPP5 34.35% 11.80% -1.38
485 MAST2 34.08% 16.10% -1.42
486 GRK6 34.04% 15.82% -1.42
487 DKFZp761P0423 33.93% 13.58% -1.44
488 VRK1 33.87% 9.28% -1.45
489 DCAMKL2 33.77% 11.96% -1.46
490 IHPK2 33.75% 9.56% -1.46
491 MATK 33.44% 10.25% -1.51
492 RP2 33.20% 13.41% -1.54
493 MAP2K2 32.98% 15.34% -1.57
494 UCK2 32.82% 13.75% -1.59
495 NEK6 32.74% 15.85% -1.60
496 PRKG1 32.52% 14.60% -1.63
497 MAP3K12 32.49% 15.33% -1.64
498 PCTK1 32.48% 13.21% -1.64
499 MGC42105 32.36% 13.85% -1.66
500 MAP2K6 32.32% 11.92% -1.66 RANK GENE AVERAGE STDEV. Z-SCORE NOTES
501 SH3BP5L 32.1 1 % 15.55% -1 .69
502 FES 32.07% 1 1.04% -1 .70
503 RKHD3 31 .89% 12.45% -1 .72
504 PRKACA 31 .76% 18.45% -1 .74 Lethal
505 MAP4K1 31 .51 % 13.69% -1 .77
506 CSNK1G1 30.32% 14.90% -1 .94
507 MAPKAPK5 30.15% 13.47% -1 .96
508 CSK 29.83% 14.1 1 % -2.01 Lethal
509 BRD3 29.16% 8.83% -2.10
510 ADRBK1 29.00% 16.93% -2.12
51 1 BRSK2 28.64% 13.09% -2.17
512 ADCK1 28.30% 13.52% -2.22
513 CSNK1G2 26.81 % 14.42% -2.43
514 CKMT1A 26.80% 12.42% -2.43
515 TSSK2 25.66% 13.30% -2.59 Lethal
516 CD2 25.01 % 14.99% -2.68
517 PIP5K1A 24.76% 15.49% -2.71
518 PHKG1 23.31 % 14.24% -2.91
519 ABL2 Low pLX DNA yield
520 ACVR2A Low pLX DNA yield
521 BLK Low pLX DNA yield
522 CDC2L1 Low pLX DNA yield
523 EGFR Low pLX DNA yield
524 EPHA3 Low pLX DNA yield
525 EPHB1 Low pLX DNA yield
526 ERBB4 Low pLX DNA yield
527 FASTKD3 Low pLX DNA yield
528 FGFR1 Low pLX DNA yield
529 FLT3 Low pLX DNA yield
530 FLT4 Low pLX DNA yield
531 HIPK3 Low pLX DNA yield
532 KSR Low pLX DNA yield
533 LYN Low pLX DNA yield
534 PANK4 Low pLX DNA yield
535 PDGFRA Low pLX DNA yield
536 PLK4 Low pLX DNA yield
537 RET Low pLX DNA yield
538 SGK3 Low pLX DNA yield
539 SRC Low pLX DNA yield
540 TXK Low pLX DNA yield
541 BRD4 Inf. Effic. < 70%
542 CAMKK2 Inf. Effic. < 70% RANK GENE AVERAGE STDEV. Z-SCORE NOTES
543 CKS1B Inf. Effic. < 70%
544 CLK3 Inf. Effic. < 70%
545 DAPK3 Inf. Effic. < 70%
546 EPHA4 Inf. Effic. < 70%
547 EPHB6 Inf. Effic. < 70%
548 FGFR3 Inf. Effic. < 70%
549 FRK Inf. Effic. < 70%
550 GCK Inf. Effic. < 70%
551 GNE Inf. Effic. < 70%
552 HIPK4 Inf. Effic. < 70%
553 ITK Inf. Effic. < 70%
554 KDR Inf. Effic. < 70%
555 LOC389599 Inf. Effic. < 70%
556 LOC649288 Inf. Effic. < 70%
557 MAP3K2 Inf. Effic. < 70%
558 NEK5 Inf. Effic. < 70%
559 NTRK1 Inf. Effic. < 70%
560 PAK1 Inf. Effic. < 70%
561 PHKG2 Inf. Effic. < 70%
562 PIK3CA Inf. Effic. < 70%
563 PIK3R3 Inf. Effic. < 70%
564 PIP5K1B Inf. Effic. < 70%
565 RIPK1 Inf. Effic. < 70%
566 RIPK2 Inf. Effic. < 70%
567 RPS6KA2 Inf. Effic. < 70%
568 RPS6KC1 Inf. Effic. < 70%
569 STK17B Inf. Effic. < 70%
570 TGFBR1 Inf. Effic. < 70%
571 UHMK1 Inf. Effic. < 70%
572 XRCC6BP1 Inf. Effic. < 70%
573 BCKDK Replicate STDEV
574 C1orf57 Replicate STDEV
575 CAMKK1 Replicate STDEV
576 CDC2L2 Replicate STDEV
577 CDK5 Replicate STDEV
578 ERBB3 Replicate STDEV
579 GSG2 Replicate STDEV
580 LOC647279 Replicate STDEV
581 LYK5 Replicate STDEV
582 MAPK1 Replicate STDEV Lethal
583 MAP2K5 Replicate STDEV
584 MAP4K2 Replicate STDEV RANK GENE AVERAGE STDEV. Z-SCORE NOTES
585 NUAK2 Replicate STDEV
586 PDK2 Replicate STDEV Lethal
587 PFKM Replicate STDEV
588 PIK3R1 Replicate STDEV
589 PLK2 Replicate STDEV
590 PRKAR2A Replicate STDEV
591 RPS6KA3 Replicate STDEV
592 RPS6KA6 Replicate STDEV
593 SGK2 Replicate STDEV
594 SRPK3 Replicate STDEV
595 VRK1 Replicate STDEV
596 VRK2 Replicate STDEV
597 VRK3 Replicate STDEV
A375 SKMEL28
Table 5: Results of a secondary screen quantifying the change in PLX4720 GI50 induced by the top 9 candidate resistance ORFs Secondary Screen
>100.
RAF1 598 2 RAF1 >10.0 >10 2
0
CRKL >10.0 59.8 3 CRKL 9.7 9.7 3
FGR >10.0 59.8 4 FGR 5 5 4
PRKCE 4.41 26.4 5 PRKCH 2.26 2.26 5
PRKCH 4.14 24.7 6 PRKCE 1.91 1.91 6
ERBB2 1.33 7.95 7 AXL 1.18 1.18 7
AXL 1 5.98 8 ERBB2 1 1 8
PAK3 0.493 2.95 9 PAK3 0.9041 0.9041 9
Controls Controls
0.152
Mock 0.91 Mock 0.5287 1.89
8
0.167
MEK1 1 (■) MEK1 1 1
1 (■) MEK DD 4.8 28 (+) MEK DD 8.29 8.29 (+) Table 6: Patient characteristics
BRAF
Time Time from status
from metastatic BRAF (on- primary disease status treatment/
Age Biopsy diagnosis diagnosis (pre- post- NRAS KRAS pMEK pERK
Patient Tissue (years) Gender Location (years) (years) Response treatment) relapse) status status status status
Tissue Partial
Pt 1 49 F Back 5 3 V600E N/A N/A N/A N/A N/A Biopsy Response
Tissue Partial
Pi 2 67 M Back 2 0 V600E N/A N/A N/A N/A N/A Biopsy Response
Tissue Partial
Pi 3 68 M Leg 11 0 V600E N/A N/A N/A N/A N/A Biopsy Response
Tissue Partial
MM-R 38 M Axilla 0 0 V600E V600E WT WT * * Biopsy Response
Short- Axillary
Stable Ref.
M307 term 59 M Lymph 4 4 V600E V600E N/A N/A **
Disease (14) Culture node
* See Figure 20
** See Figure 3f

Claims

1 . A method of identifying a subject having cancer who is likely to benefit from treatment with a combination therapy with a RAF inhibitor and a second inhibitor, comprising:
(a) assaying a gene copy number, a mRNA or a protein level or
phosphorylation of one or more kinase targets selected from the group consisting of MAP3K8 (TPL2/COT), RAF1 (CRAF), CR L(CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3) in cancer cells obtained from the subject and comparing the gene copy number, the mRNA or the protein level or the phoshorylation with a gene copy number, a mRNA or a protein level or phosphorylation of the target kinase in cells obtained from a subject without the cancer; and
(b) correlating increased gene copy number or an alteration in mRNA expression or protein overexpression or phosphorylation of the target kinase in the cancer cells relative to the cells from the subject without the cancer with the subject having the cancer who is likely to benefit from treatment with the combination therapy.
2. The method of claim 1 , further comprising (c) assaying a nucleic acid sample obtained from the cancer cells for the presence of a mutation in a nucleic acid molecule encoding a B-RAF polypeptide with a mutation at about amino acid position 600 and (d) correlating the presence of the mutation in the nucleic acid molecule encoding the B-RAF polypeptide with a subject who is likely to benefit from treatment with the combination therapy.
3. The method of claim 1 , comprising correlating the presence of a Valine to Glutamic Acid mutation at about amino acid position 600 (V600E) in a nucleic acid molecule encoding B-RAF with a subject who is likely to benefit from treatment with the combination therapy.
4. The method of claim 1 , comprising assaying the gene copy number, the mRNA or the protein level of MAP3K8 (TPL/COT).
5. The method of claim 1 , comprising assaying phosphorylation of C-RAF at amino acid S338.
6. The method of claim 1 , wherein the second inhibitor is a MEK inhibitor, a CRAF inhibitor, a CrkL inhibitor or a TPL2/COT inhibitor.
7. The method of claim 1 , wherein the RAF inhibitor is a B-RAF inhibitor or a pan-RAF inhibitor.
8. The method of claim 1 , wherein the RAF inhibitor is selected from the group consisting of RAF265, sorafenib, SB590885, PLX 4720, PLX4032, GDC-0879 and ZM 336372.
9. The method of claim 1 , wherein the subject has innate resistance to the RAF inhibitor or is likely to develop resistance to the RAF inhibitor.
10. The method of claim 1 , wherein the cancer is selected from the group consisting of melanoma, breast cancer, colorectal cancers, glioma, lung cancer, ovarian cancer, sarcoma and thyroid cancer.
1 1 . The method of claim 10, wherein the cancer is melanoma.
12. A method of treating cancer in a subject in need thereof, comprising administering to the subject an effective amount of a RAF inhibitor and an effective amount of a second inhibitor, wherein the second inhibitor is a MEK inhibitor, or a MAP3K8 (TPL2/COT) inhibitor.
13. The method of claim 12, wherein the subject has cancer cells comprising a mutation in B-RAF.
14. The method of claim 12, wherein the subject has cancer cells comprising a B- RAFV6OOE M UTATION_
15. The method of claim 12, further comprising assaying a gene copy number, a mRNA or a protein level or phosphorylation of one or more kinase targets selected from the group consisting of MAP3K8 (TPL2/COT), RAF1 (CRAF), CR L(CrkL), FGR (Fgr), PRKCE (Prkce), PRKCH (Prkch), ERBB2 (ErbB2), AXL (Axl), or PAK3 (Pak3) in cancer cells obtained from the subject and comparing the gene copy number, the mRNA or the protein level or the phoshorylation with a gene copy number, a mRNA or a protein level or phosphorylation of the target kinase in cells obtained from a subject without the cancer; and
administering to the subject the effective amount of a RAF inhibitor and the effective amount of a second inhibitor in subjects having an increased gene copy number or an alteration in mRNA expression or protein overexpression or phosphorylation of the target kinase in the cancer cells.
16. The method of claim 12, wherein the RAF inhibitor is selected from the group consisting of RAF265, sorafenib, SB590885, PLX 4720, PLX4032, GDC-0879 and ZM 336372.
17. The method of claim 12, wherein the MEK inhibitor is selected from the group consisting of CI-1040/PD184352, AZD6244, PD318088, PD98059, PD334581 , RDEA1 19, 6-Methoxy-7-(3-morpholin-4-yl-propoxy)-4-(4-phenoxy-phenylamino)- quinoline-3-carbonitrile and 4-[3-Chloro-4-(1 -methyl-1 H-imidazol-2-ylsulfanyl)- phenylamino]-6-methoxy-7-(3-morpholin-4-yl-propoxy)-quinoline-3-carbonitrile.
18. The method of claim 12, wherein the subject has innate resistance to the RAF inhibitor or is likely to develop resistance to the RAF inhibitor.
19. The method of claim 12, wherein the cancer is selected from the group consisting of melanoma, breast cancer, colorectal cancers, glioma, lung cancer, ovarian cancer, sarcoma and thyroid cancer.
20. The method of claim 12, wherein the cancer is melanoma.
21 . The method of claim 12, wherein the subject has innate resistance to the MEK inhibitor or is likely develop resistance to the MEK inhibitor.
22. The method of claim 12, wherein the second inhibitor is a MAP3K8
(TPL2/COT) inhibitor.
23. A method of identifying a kinase target that confers resistance to a first inhibitor, the method comprising:
culturing cells having sensitivity to the first inhibitor;
expressing a plurality of kinase ORF clones in the cell cultures, each cell culture expressing a different kinase ORF clone;
exposing each cell culture to the inhibitor;
identifying cell cultures having greater viability than a control cell culture after exposure to the inhibitor to identify one or more kinase ORF clones that confers resistance to the first inhibitor.
24. The method of claim 23, wherein the cultured cells have sensitivity to a RAF inhibitor.
25. The method of claim 23, wherein the cultured cells have sensitivity to a MEK inhibitor.
26. The method of claim 23, wherein the cultured cells comprise a B-RAF mutation.
27. The method of claim 26, wherein the cultured cells comprise a B-RAFV600E mutation.
28. The method of claim 23, wherein the cultured cells comprise a melanoma cell line.
29. The method of claim 23, comprising providing the plurality of kinase ORF clones in a lentiviral vector or a retroviral vector.
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